Cellular extracts

ABSTRACT

The invention describes methods and agents for improving cosmetic appearance, for promoting, improving or restoring health of cells and tissues, preferably skin, and more preferably, for restoring aged or damaged skin to a healthy appearance. The agents include compositions of cells, eggs, cell extracts, egg extracts, and extract components such as purified nucleic acids, polypeptides, lipids, carbohydrates or other natural products.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/131,198, filed Apr. 18, 2016, now U.S. Pat. No. 9,999,639, which is acontinuation of U.S. patent application Ser. No. 14/526,751, filed Oct.29, 2014, now U.S. Pat. No. 9,314,488, which is a divisional of U.S.patent application Ser. No. 12/437,100, filed May 7, 2009, now U.S. Pat.No. 8,877,253, which claims the benefit of U.S. Prov. Appl. 61/051,931filed May 9, 2008 and U.S. Prov. Appl. 61/120,146 filed Dec. 5, 2008,and is a continuation-in-part of U.S. application Ser. No. 11/801,778,filed May 11, 2007, now U.S. Pat. No. 8,075,920, which claims thebenefit U.S. Prov. Appl. 60/799,560, filed May 11, 2006, of all of whichare incorporated herein by reference in their entirety.

FIELD OF THE INVENTION

The invention relates to the use of cellular extracts to delivercompounds in a topical fashion, the use of cellular extracts to increaseor decrease expression of genes, and the production of cellularextracts, especially from fish and amphibian eggs.

BACKGROUND OF THE INVENTION

Skin is the first barrier a human has against outside aggressions, andcarries out both physical and chemical defenses. Vitamin D is producedin the epidermis under the effects of solar radiation. This vitamin isnecessary for calcium to be absorbed in the intestine and then fixed onthe bones, which enables the development and growth of the human body.However, excessive sun exposure leads to skin damage and potentiallycancer. In addition, skin cells may become damaged by physical means,i.e., wounded, or damaged due to age. Thus, there is a need to identifycompositions and methods for managing and improving skin health andpreventing and treating skin conditions, and diseases, and maintainingnormal skin appearance and restoring aged skin to a youthful appearance.

When a wound heals, a scar takes its place. Simple tissues such as fat,connective tissue, and epithelium regenerate, but the skin, being acomplex organ derived from 2 germ layers, heals by the formation of apredominantly fibrous tissue. If the injury sections or destroys thepapillary layer of the stratum corneum, a scar will be formed.Sometimes, this scar is inconspicuous; other times, it may bedisfiguring. The most common presenting concern of patients withabnormal scars is disfigurement. However, some patients experience othersymptoms in association with their abnormal scar, such as pain, puritis,and loss of motion. These other symptoms can be indications for surgicalcorrection of the scar. Thus, there is a need to identify compositionsand methods of managing, preventing, and treating scars.

After damage, surgery or radiation of tissues or organs, scarring andincomplete regeneration of the tissue or organ occurs. This causes painand discomfort as well as compromised function of the damaged tissues ororgans. Generally, adult humans are unable to completely heal and regroworgans and tissues, while e.g. amphibians can regrow tissues includinglost limbs. Thus, there is a need to identify compositions and methodsof managing and treating scarring in internal organs and tissues,increasing tissue plasticity and stimulate regrowth of damaged tissuesand organs.

SUMMARY OF THE INVENTION

The invention relates to improving visible parts of a personcontributing to cosmetic appearance directly or indirectly, includingbut not limited to skin, hair, subcutaneous fat, cartilage, muscle,skeletal structures, and to improve health and damage of cells andtissues preferably skin, and more preferably restoring aged skin to ayouthful appearance. In some embodiments, the invention relates tocompositions of cells, cell or egg extracts, and extract componentswhich can induce de-differentiation, including but not limited topurified or synthetic nucleic acid sequences, polypeptides, or naturalproducts contained in the extracts. In some embodiments, the cells aredifferentiable cells, preferably stem cells. In some embodiments, thecompositions are used in a method that comprises application of thecompositions to skin and/or wounds after removal the outer surfacelayers. In some embodiments, the invention relates to a method ofde-differentiation of cells and/or de-differentiation followed byre-differentiation. In some embodiments, the invention relates tomanaging, preventing, and treating skin diseases. In some embodiments,the invention relates to repair or de novo formation of lost or damagedtissues, organs and limbs.

In some embodiments, the invention relates to a composition comprisingi) a cellular component comprising differentiable cells, differentiablecell cellular extracts, egg cellular extracts or components ofdifferentiable cell extracts or egg cellular extracts or combinationsthereof and ii) lipids. In further embodiments, the composition furthercomprises purified or synthetic nucleic acid sequences, proteins,epigenetic inhibitors, or natural products contained in the extracts orcombinations thereof. In further embodiments, the differentiable cellsare embryonic stem cells, embryonic germ cells, or adult stem cells. Thepresent invention is not limited to the use of any particular cellularextract or fraction. Indeed, the use of a variety of cellular extractsand fractions is contemplated, including, but not limited to,cytoplasmic extracts and fractions, nuclear extracts and fractions,water soluble extracts and fractions, and extracts and fractionsprepared from cellular extracts by affinity chromatography, gradientcentrifugation, HPLC, size exclusion chromatography and the like.

In some embodiments, the invention provides methods and the compositionsfind use for prevention of deterioration, damage and malfunction ofcells and tissues, and to promote, improve and exceed cellular functionin order to promote, improve and exceed appearance, vitality and healthof cells and tissues.

In some embodiments, the invention provides a skin healing compositioncomprising i) a cellular component selected from the group consisting ofdifferentiable cells, differentiable cell cellular extracts and an eggcellular extract or combinations thereof and ii) a lipid component. Infurther embodiments, the synthetic protein is a fusion-trojan protein.In further embodiments, the composition further comprises naturalvernix, vernix extracts, vernix made from synthetic components, andcomponents of vernix extracts. In further embodiments, the lipidcomponent comprises squalene, aliphatic waxes, sterol esters, diolesters, triglycerides, and free sterol. In some embodiments, the lipidcomponent is from a source other than the source of the extract, e.g., apurified lipid from a different source, either natural or synthetic. Infurther embodiments, the lipid component is derived from egg from fish,shrimp, sea urchin or frog and/or fish roe. In further embodiments, thelipid component contains cholesterol, fatty acids, and ceramides. Insome embodiments, the lipid component is from a source different thanthe cellular component. In further embodiment, the composition containskeratin or filaggrin. In further embodiments, the composition furthercomprises glutamine, anti-infective agents, antioxidants and/ornicotinamide. In further embodiments, the antioxidant is vitamin E, A,or C or combinations thereof.

In some embodiments the invention provides a kit for improving theappearance of a scar comprising two compositions, wherein the firstcomposition dissolves scar tissue and comprises collagen dissolvingagents and the second composition improves wound healing and comprises acellular component selected from the group consisting of differentiablecells, differentiable cell cellular extracts and an egg cellular extractor combinations thereof, lipids, proteins, and water. In furtherembodiments, the differentiable cells are embryonic stem cells,embryonic germ cells, or adult stem cells. In further embodiments, thefirst composition further comprises an antiseptic compound, anantibacterial compound, an anti-inflammatory compound, animmunomodulator, a protease, or an analgesics or combinations thereof.In further embodiments, the second composition further comprises naturalvernix, vernix extracts, vernix made from synthetic substances, andcomponents of vernix extracts. In further embodiments the lipidcomponent comprises squalene, aliphatic waxes, sterol esters, diolesters, triglycerides, free sterols or combinations thereof. In furtherembodiments, the lipids and/or proteins are derived from eggs from fish,shrimp, sea urchin or frog and/or fish roe. In further embodiments, thelipid fraction contains cholesterol, fatty acids, or ceramides or acombination thereof. In some embodiments, the lipid component is from adifferent source than the cellular component. In further embodiments,the composition further comprises glutamine, antiinfective agents,antioxidants and/or nicotinamide.

In some embodiments, the invention provides methods for improving theappearance of a skin comprising: i) removing skin tissue by chemicals, alaser, or physical force and ii) applying a composition that improveswound healing comprising differentiable cells, differentiable cell oregg cellular extracts, components of differentiable cell extracts,lipids, proteins, and/or water. In further embodiments, improving theappearance of skin includes improving the appearance of a scar orimproving the appearance of skin with wrinkles. In further embodiments,the differentiable cells are embryonic stem cells, embryonic germ cells,or adult stem cells. In further embodiments, the composition furthercomprises natural vernix, vernix extracts, vernix made from syntheticsubstances, and components of vernix extracts.

In additional embodiments, the invention provides methods for thetopical administration of differentiable cells, egg or differentiablecell cellular extracts, components of cell extracts comprising:providing a composition comprising a cellular component comprisingdifferentiable cells, egg or differentiable cell cellular extracts,components of cell extracts and a subject having skin and applying theextracts to the skin of the subject. In further embodiments, the egg ordifferentiable cellular extracts or components of cell extracts areeffective as a nutrient to a cell of the skin. In further embodiments,the composition is a water-based gel. In further embodiments, thewater-based gel comprises a compound selected from the group consistingof hyaluronic acid and chitosan. In further embodiments the compositionis a component on a wound dressing. In further embodiments thecomposition is a component in a spray composition. In furtherembodiments the spray composition is an aerosol. In further embodiments,the spray composition dries on the skin. In further embodiments, thespray composition comprises gel-forming components. In some embodiments,the composition further comprises a lipid component as described above.

In some embodiments, the invention provides a wound healing dressingcomprising a composition comprising differentiable cells, egg ordifferentiable cell cellular extracts, and components of cell extracts.

In additional embodiments, the invention provides methods for thetopical administration of differentiable cells, cell extracts, andcomponents of cell extracts comprising: i) providing a) a compositioncontaining differentiable cells, differentiable cell or egg cellularextracts, components of cell extracts, b) a subject having a wound inskin and c) wound dressing ii) applying the differentiable cells, cellextracts, components of cell extracts to the wound; and iii) coveringthe wound with the wound dressing. In further embodiments, the wounddressing is non-occlusive. In further embodiments, the wound dressing isplaster. In further embodiments, the wound dressing comprises: i) awaterproof layer; ii) a nutrient gel layer comprising differentiablecells, cell extracts, and components of cell extracts. In furtherembodiments, the waterproof layer is a polymeric (i.e., plastic)membrane that can be glued onto skin. In further embodiments, thenutrient gel layer comprises antibacterial agents and collagenmodulating substances. In further embodiments, the nutrient gel layerimproves the speed of wound healing.

In some embodiments, the invention provides methods for the topicaladministration of differentiable cells, egg or differentiable cellcellular extracts, or components of cell extracts comprising: i)providing a) a subject having 1) a wound in skin and 2) a tissuecomprising specialized cells b) wound dressing; ii) harvesting thespecialized cells from the tissue; iii) culturing the specialized cellsunder conditions such that a composition comprising the culturedspecialized differentiable cells, cell extracts, or components of cellextracts is formed; iii) applying the composition to the wound; and iv)covering the wound with the wound dressing. In further embodiments, thespecialized cells selected from the group consisting of a bulgehair-follicle stem cell, an embryonic stem, or germ stem cell. Infurther embodiments, the composition is a fluid suspension ofspecialized cells. In further embodiments, the composition is a plaster.In further embodiments, the composition is placed on a membrane with anutrient gel layer prior to applying the composition to the wound. Infurther embodiments, the membrane is polymeric (i.e., plastic)functioning as an occlusive wound dressing when applied to the skin. Infurther embodiments, the wound dressing is a commercial band-aid. Infurther embodiments, prior to applying the composition a step of burningskin is performed, freezing skin is performed, and/or sanding skin isperformed. In further embodiments, prior to applying the composition atransport vehicle which penetrate intact skin is applied to thecomposition or skin comprising a phospholipids, palmitylmyristrates,DMSO, polymer or chitosan suspensions or matrix, liposomes and/or trojanpeptides, chariot peptides, small elastic vesicles (Van den Bergh etal., 1999), microspheres, nanoparticles, preloaded spherical beads, uni-and/or multilamellar vesicles, retinol molecular film, poly acrylonitrile, beta-glucan (Redmond, Int. J. Cosmetic Science 2005), propyleneglycol, butylenes glycol, polyethylene glycol, olive oil, dimethylisosorbate, dimethylformamide, methyl salicylate, long chain oleicacids.

In some embodiments, the invention provides compositions for stimulatingcells such as fibroblasts and keratinocytes comprising an effectiveamount of a purified cytoplasmic fraction of an embryonic stem cell,progenitor cell, somatic cell or eggs from animals, including but notlimited to primates, rodents, fish, shrimp, sea urchin and/or frog egg.In further embodiments, the composition further comprises fats, proteinsand/or natural products. In further embodiments, the composition furthercomprises an herbal substance. In further embodiments, the herbalsubstance is aloe vera. In further embodiments, the composition furthercomprises seed extracts. In further embodiments, the seed extracts areobtained from wheat, corn, rice, or avocado. In further embodiments, thecomposition further comprises a plant oil. In further embodiments, thecomposition further comprises a fungal substance. In furtherembodiments, the fungal substance is nepal fungus. In furtherembodiments, the composition further comprises fish, shrimp, sea urchin,or frog egg extracts, or components of these egg extracts. In furtherembodiments the components of egg extracts are glycosylation breakersand inhibitors. In further embodiments, the components of egg extractsare glycosylation breakers and inhibitors are aminoguanidine, carnosine,and fex pyridoxamine.

In additional embodiments, the invention provides methods of woundhealing comprising providing a subject having a wound and a compositioncomprising differentiable cells, differentiable cell or egg cellularextracts, egg extracts, components of cell extracts or egg extracts andapplying the composition to the wound under conditions such that thewound is healed. In further embodiments, the composition furthercomprises a collagen dissolving agent. In further embodiments, thecollagen dissolving agent is an acid. In further embodiments, thecomposition further comprises a fruit acid. In further embodiments thecomposition is a cream. In further embodiments, the wound is an openwound and applying the composition topically. In preferred embodiments,the method further comprises providing a support matrix wherein, thesupport matrix comprises the composition. In further embodiments, thesupport matrix is a fabric or plastic wound dressing.

In some embodiments, the invention provides methods of skin regenerationcomprising providing a subject having a wound and a compositioncomprising differentiable cells, differentiable cell or egg cellularextracts, or components of cell extracts or egg extracts and applyingthe composition to the wound under conditions that such skin isregenerated. In further embodiments the composition is a cream. Infurther embodiments, the wound is an open wound and the composition isapplied topically.

In additional embodiments, the invention relates to a method of skinrejuvenation comprising providing a subject having an uneven skin and acomposition comprising differentiable cells, differentiable cell or eggcellular extracts, egg extracts, or a component of a cellular extractand applying the composition to the uneven skin under conditions thatsuch skin is rejuvenated. In some embodiments, the component of a cellextract is a nucleic acid sequence or the component of a cell extract isa peptide or combinations thereof. In some embodiments, the uneven skinis a result of a scar or wrinkles. In further embodiments, thecomposition is in a cream. In further embodiments the cream furthercomprises permeabilizing agents. In further embodiments, thepermeabilizing agent is a toxic agent, DMSO or chitosan, chitosanpolymer, or trypsin. In further embodiments, the permeabilizing agent isliposomes or alginate beads. In further embodiments, the liposomes oralginate beads comprise a peptide or a nucleic acid sequence of a cellextract or growth factor or a combination thereof. In furtherembodiments, the liposome comprises nucleic acid sequence of cellextracts or egg extracts generated by electroporation. In furtherembodiments, the composition comprises a fusion trojan peptidecomprising a peptide of the cell extract. In further embodiments, thecomposition is applied topically. In additional embodiments, the methodfurther comprises the step of applying the composition is executed afterapplying a chemical, laser, or physical force to the uneven skin underconditions that an outer lay of cells of the uneven skin are removed. Infurther embodiments, the composition further comprises an antisepticcompound, an antibacterial compound, an anti-inflammatory compound, animmunomodulator, a protease, or an analgesic compound or combinationsthereof.

In some embodiments, the invention relates to a composition comprising:a lipid; a composition of plant seed components; an antioxidant; apurified or synthetic protein, or a purified or synthetic naturalproduct contained in a cellular extract; a stabilizing component;autologous fat derived from adipose tissue of a subject.

In additional embodiments, the invention provides methods of improving askin graft comprising grafting skin or skin substitute and applying acomposition comprising: differentiable cells, differentiable cell or eggcellular extracts, egg extracts; components of cell extracts or eggextracts; a purified or synthetic nucleic acid sequence, a purified orsynthetic protein, or a purified or synthetic natural product contain incell extracts, egg extracts; or combinations thereof.

In some embodiments, the invention provides methods for managing,treating, and/or preventing scarring, abnormal scars, abnormal woundhealing, widened scar, hypertrophied scar, keloid, keloid scar,wound-healing complications, cicatrix, and/or scar hypertrophy byadministering in a prophylactic or non-prophylactic manner thecompositions disclosed herein. In further embodiments, the inventionprovides methods for primary healing, wound closure, secondary healing,epithelialization, reepithelialization, tertiary wound closure, delayedprimary closure, debridement, and suture using the compositionsdescribed herein. In other embodiments, the compositions describedherein are used to increase or decrease at the site of administration toa subject, inflammatory phase, proliferative phase, maturational phase,hemostasis, inflammation, collagen, clotting, thromboxane A2,prostaglandin 2a, prostaglandin 2-alpha, vasoconstrictor, hemorrhage,vasodilatation, histamine, platelet, chemokine, epidermal growth factor,fibronectin, fibrinogen, histamine, platelet derived growth factor,serotonin, von Willebrands factor, clot formation, plateletdegranulation, complement cascade, neutrophil, leukocyte, macrophage,monocyte, collagenase, interleukin, tumor necrosis factor, fibroblasts,transforming growth factor, keratinocyte, angiogenesis, granulationtissue formation, collagen deposition, and insulin-like growth factor.

In some embodiments, the invention provides compositions comprisingdifferentiable cells, preferably embryonic stem cells or precursorcells. In further embodiments, the compositions comprise the extracts ofdifferentiable cells, preferably embryonic stem cells or precursorcells. In additional embodiments, the compositions contain components ofextracts from differentiable cells, preferably embryonic stem cells orprecursor cells.

In some embodiments, the invention provides compositions containingdifferentiable cells, preferably embryonic stem cells or precursorcells, the extracts of differentiable cells, preferably embryonic stemcells or precursor cells, components of extracts from differentiablecells, and/or natural vernix and/or vernix extracts and/or vernixcomponents of vernix extracts that partially or totally synthetic.

In some embodiments, the invention provides methods for the topicaladministration of egg cellular extracts or differentiable cell cellularextracts comprising: providing a composition containing egg cellularextracts or differentiable cell cellular extracts and a subject havingskin and applying the extracts to the skin. Preferably a nutritionalsignal in the extract reaches and is effective as a nutrient to the skincells. Preferably the composition is in a water based gel comprisinghyaluronic acid and/or chitosan. In another preferred embodiment, theextract is a spray acting as a liquid band-aid or fluid that dries onthe skin. In further embodiments, the liquid contains gel-formingcomponents such as collagen and chitosan. In further preferredembodiments, the composition is a component of a film on a support orcream.

The present invention also provides for use of the foregoingcompositions for the treatment of skin, for removing wrinkles, forrejuvenation of skin, for wound healing, for improving the appearance ofskin, the prevent damage to skin, to prevent deterioration of skin, orto provide nutrients to skin and any other use described herein.

The present invention further provides methods for preparing acomposition for topical application to the skin comprising: providingdifferentiable cells or preparing an extract or fraction ofdifferentiable cells or eggs; and formulating said differentiable cellsor said extract with an agent for topical administration to the skin toprovide a cream, gel, spray, emulsion, solid, plastic or matrix,ointment, powder or lotion suitable for topical administration. Infurther embodiments, the present invention provides compositions made bythe foregoing methods.

In further embodiments, the present invention provides compositionscomprising: an egg cellular extract; and a purified active compound. Insome embodiments, the purified active compound is selected from thegroup consisting of a small molecule pharmaceutical compound and apharmaceutical protein. In some embodiments, the egg cellular extract isselected from the group consisting of an extract of an activated fishegg, an unactivated fish egg, an activated amphibian egg and anunactivated amphibian egg. In some embodiments, the egg extract is froma fertilized egg. In some embodiments, the egg extract if from an eggselected from the group consisting of an amphibian egg and a fish egg.In some embodiments, the composition is provided in a cream, gel,emulsion, ointment, spray, powder or lotion. In some embodiments, theegg extract is a cytoplasmic extract.

In some embodiments, the methods of increasing the penetration of anactive compound into the skin of a subject comprising: a) providing thepreceding composition and b) topically applying the composition to theskin of the subject so that the active compound is absorbed into theskin of the subject.

In some embodiments, the present invention provides methods ofincreasing collagen protein production and/or collagen gene expressionby the skin of a subject, comprising: providing a composition comprisingan egg extract; and applying the egg extract to the skin of the subjectunder conditions such that collagen protein production and/or collagengene expression is increased at the site of application. In someembodiments, the composition is applied to a wound in the skin of thesubject. In some embodiments, the egg cellular extract is selected fromthe group consisting of an extract of an activated fish egg, anunactivated fish egg, an activated amphibian egg and an unactivatedamphibian egg. In some embodiments, the egg extract is from a fertilizedegg. In some embodiments, the egg extract if from an egg selected fromthe group consisting of an amphibian egg and a fish egg. In someembodiments, the composition is provided in a cream, gel, emulsion,ointment, spray, powder or lotion. In some embodiments, the egg extractis a cytoplasmic extract.

In some embodiments, the present invention provides methods ofincreasing cell proliferation in the skin of a subject, comprising:providing a composition comprising an egg extract; and applying the eggextract to the skin of the subject under conditions such that cellproliferation is increased at the site of application. In someembodiments, the composition is applied to a wound in the skin of thesubject.

In some embodiments, the present invention provides methods of preparingextracts from amphibian of fish eggs comprising: a) providing eggs fromselected from the group consisting of amphibian and fish egg b) treatingthe eggs to prevent growth of bacteria during the further processing ofthe eggs. In some embodiments, the eggs are treated with a bactericidalor bacteristatic composition. In some embodiments, the compositioncomprises iodine.

In some embodiments, the present invention provides methods of makingextracts from amphibian of fish eggs comprising: a) providing eggs fromselected from the group consisting of amphibian and fish eggs; b)processing the eggs to provide a cytoplasmic fraction from the eggs; andc) treating the cytoplasmic fraction to prevent the growth of bacteria.In some embodiments, the eggs are selected from the group consisting offertilized and unfertilized eggs. In some embodiments, the processingcomprising homogenization of the eggs followed by centrifugation toseparate lipids and solids from the cytoplasmic fraction. In someembodiments, the treating comprising heating the extract to atemperature of from about 50 C to about 90 C for a period of greaterthan one minute.

In some embodiments, the present invention provides compositionscomprising about 100 to 250 mg/ml fish egg protein in an aqueoussolution; at least one trace element selected from the group consistingof calcium, phosphorous, zinc, copper and iron; at least one vitaminselected from the group consisting of vitamins A, E, riboflavin, niacin,B 6, calcium pantothenate and B 12; and a lipid fraction comprising fromabout 60 to 80% unsaturated fatty acids; wherein the composition has anosmolarity of from about 330 to 440 mOsm, a pH of from about 5.0 to 7.7,and density of from about 0.8 to 1.4 g/ml.

In some embodiments, the present invention provides methods ofincreasing collagen protein production and/or collagen gene expressionby the skin of a subject, comprising: applying a composition comprisinga fish egg cellular extract comprising about 50 to 500 mg/ml fish eggprotein, from about one to six grams/100 grams extract fish egg lipids,an osmolarity of from about 330 to 440 mOsm, and a pH of from about 5.0to 7.7 to the skin of the subject under conditions such that collagenprotein production and/or collagen gene expression is increased at thesite of application. In some embodiments, the composition is applied toa wound in the skin of the subject. In some embodiments, the compositionis provided in a cream, gel, emulsion, ointment, spray, powder orlotion.

In some embodiments, the present invention provides methods of improvinga parameter of wound healing in a subject, comprising: applying acomposition comprising a fish egg cellular extract comprising about 50to 500 mg/ml fish egg protein, from about one to six grams/100 gramsextract fish egg lipids, an osmolarity of from about 330 to 440 mOsm,and a pH of from about 5.0 to 7.7 to a wound of the subject underconditions such that a parameter of wound healing is improved, whereinthe parameter is selected from the group consisting of faster drying,faster reepithelialization, reduced inflammation, faster contraction,earlier remodeling, improved remodeling, reduction in scar tissue andimproved visual appearance of the wound and combinations thereof. Insome embodiments, the composition is applied to a wound in the skin ofthe subject. In some embodiments, the composition is provided in acream, gel, emulsion, ointment, spray, powder or lotion. In someembodiments, the egg extract is a cytoplasmic extract.

In some embodiments, the present invention provides methods ofincreasing expression of a gene in a tissue of a subject comprisingcontacting the tissue of the subject with an egg extract underconditions such that expression of a gene is increased, wherein the geneis selected from the group consisting of collagen 1, collagen 3, VEGF-B,VEGF-C, TGFβ2, TGFβ3, PDGF-A, PDGF-B, PDGF-D, IL-18, and fibronectin. Insome embodiments, the egg extract is a cytoplasmic extract of fish oramphibian eggs.

In some embodiments, the present invention provides methods ofdecreasing expression of a gene in a tissue of a subject comprisingcontacting the tissue of the subject with an egg extract underconditions such that expression of a gene is decreased, wherein the geneis selected from the group consisting of a matrix metallopeptidase,TGFβ1, VEGF-A, elastin, IL 1β, and IL 12. In some embodiments, the eggextract is a cytoplasmic extract of fish or amphibian eggs. In someembodiments, the matrix metallopeptidase (MMP) is selected from thegroup consisting of MMP 14, 16, 17, 19, 20, 23, 25 and 28.

In some embodiments, the present invention provides methods of treatinga subject with skin condition comprising contacting the skin of thesubject with a fish or amphibian cytoplasmic egg extract in an effectiveamount, wherein the skin condition is selected from the group consistingof ulcers, psoriasis, calluses, moles, acne, rosacea, dermatitis,keratosis, basal cell carcinoma and squamous cell carcinoma. In someembodiments, the fish or amphibian cytoplasmic cell extract is providedin a cream, gel, emulsion, ointment, spray, powder or lotion.

DESCRIPTION OF THE FIGURES

FIG. 1 is a graph of generations over time.

FIG. 2 is a graph of minutes/generation v. time.

FIG. 3 is a growth curve graph.

FIG. 4 provides graphs of mice skin wound and scar measurements andwound healing rates. These data show that the wound healing extract hasan effect on healing of two types of wounds (excision wounds leftpanels, incision wounds rights panels) in mouse skin. Measures taken byruler and wound/scar area traced on transparent film at day 1, 5, 9 and12. Areas of excision and length of incision wounds (top panels) show agradual reduction in wound area from day 1 to 12. The healing startsearlier and the wound reduction is more rapid in extract treatedanimals, significant at day 5 and 9 for excision wounds. Scars formedwere measured from day of reepithelization (middle panels). A tendencyto smaller wound sizes is seen for both excision and incision wounds.Day of complete healing taken as day scab falls wound is revealed. Thepercent of animals with completely healed wounds (bottom panels) showmore rapid healing in the treated animals for the incision wounds.

FIG. 5 provides a graph demonstrating the effect of LEX on proliferationof fibroblasts in vitro. Diamond—control, square—LEX6, triangle—LEX15.

FIG. 6 provides a graph of the fold induction of trout roe, unfertilizedsalmon egg (salmon roe) and fertilized salmon egg (eyeroe) extracts.

DEFINITIONS

“Anti-infective agents” include, but are not limited tobenzylpenicillin, penicillin, penicillin G, 6-phenyl acetyl penicillin,penicillin V, micronomicin, clavulanate, oxacillin, dequalinium,cloxacillin, sulbenicillin, ampicillin, cilleral, and principen andcombinations thereof.

“Anti-inflammatory” means a substance that reduces inflammation. Manyanalgesics remedy pain by reducing inflammation. Manysteroids—specifically glucocorticoids—reduce inflammation by binding tocortisol receptors. Non-steroidal anti-inflammatory drugs (NSAIDs)alleviate pain by counteracting the cyclooxygenase (COX) enzyme. On itsown COX enzyme synthesizes prostaglandins, creating inflammation. Manyherbs have anti-inflammatory qualities, including but not limited tohyssop and willow bark (the latter of which contains salicylic acid, theactive ingredient in aspirin), as well as birch, licorice, wild yam andginseng.

“Antioxidants” means any of a variety of substances that prevent or slowthe breakdown of another substance by oxygen. Synthetic and naturalantioxidants are used to slow the deterioration of gasoline and rubber,and such antioxidants as vitamin C (ascorbic acid), butylatedhydroxytoluene (BHT), and butylated hydroxyanisole (BHA) are typicallyadded to foods to prevent them from becoming rancid or from discoloring.Nutrients such as beta-carotene (a vitamin A precursor), vitamin C,vitamin E, and selenium have been found to act as antioxidants. They actby scavenging free radicals, molecules with one or more unpairedelectrons, which rapidly react with other molecules, starting chainreactions in a process called oxidation. Free radicals are a normalproduct of metabolism; the body produces its own antioxidants (e.g., theenzyme superoxide dismutase) to keep them in balance. However, stress,aging, and environmental sources such as polluted air and cigarettesmoke can add to the number of free radicals in the body, creating animbalance. The highly reactive free radicals can damage healthy DNA andhave been linked to changes that accompany aging (such as age-relatedmacular degeneration, a leading cause of blindness in older people) andwith disease processes that lead to cancer, heart disease, and stroke.

An “antiseptic” is a substance that kills or prevents the growth andreproduction of various microorganisms, including bacteria, fungi,protozoa, and viruses on the external surfaces of the body. Theobjective of antiseptics is to reduce the possibility of sepsis,infection or putrefaction by germs. Antibacterials have the sameobjective but only act against bacteria. Antibiotics perform a similarfunction, preventing the growth or reproduction of bacteria within thebody. Antiseptics include, but are not limited to, alcohol, iodine,hydrogen peroxide, and boric acid. There is great variation in theability of antiseptics to destroy microorganisms and in their effect onliving tissue. For example, mercuric chloride is a powerful antiseptic,but it irritates delicate tissue. In contrast, silver nitrate killsfewer germs but can be used on the delicate tissues of the eyes andthroat. There is also a great difference in the time required fordifferent antiseptics to work. Iodine, one of the fastest-workingantiseptics, kills bacteria within 30 sec. Other antiseptics haveslower, more residual action. Since so much variability exists, systemshave been devised for measuring the action of an antiseptic againstcertain standards. The bacteristatic action of an antiseptic compared tothat of phenol (under the same conditions and against the samemicroorganism) is known as its phenol coefficient.

“Chitosan” is a beta-1,4-linked glucosamine polymer which, unlikechitin, contains few, if any, N-acetyl residues. It may be obtained fromchitin, a polysaccharide found in the exoskeletons of crustaceans suchas shrimp, lobster, and crabs. The shells may be ground into a pulverouspowder. This powder is then deacetylated which allows the chitosan toabsorb lipids.

“Collagen” means any of a variety of substances that contains the alphachains of the collagen polypeptide with a sequence that generallyfollows the pattern Gly-X-Y, where Gly for glycine, X for proline, and Yfor proline or hydroxyproline. Collagen proteins also containsignificant amounts of glycine and proline. Hydroxyproline andhydroxylysine are not inserted directly by ribosomes. They arederivatized from proline and lysine in enzymatic processes ofpost-translational modification, for which vitamin C is required. Thisis related to why vitamin C deficiencies can cause scurvy, a diseasethat leads to loss of teeth and easy bruising caused by a reduction instrength of connective tissue due to, a lack of collagen, or defectivecollagen. Cells called fibroblasts form the various fibers in connectivetissue in the body including collagen. The white collagen that makes upthe matrix of most connective tissue in mammals consists of inter-wovenfibers of the protein collagen. The collagen fibers consist of globularunits of the collagen sub-unit, tropocollagen. Tropocollagen sub-unitsspontaneously arrange themselves under physiological conditions intostaggered array structures stabilized by numerous hydrogen and covalentbonds. Tropocollagen sub-units are left-handed triple helices where eachstrand is, further, a right-handed helix itself. Thus, tropocollagen maybe considered to be a coiled coil.

Although collagen is responsible for skin elasticity, and itsdegradation leads to wrinkles that accompany aging, it occurs in manyother places throughout the body, and in different forms known as types:Type I collagen—This is the most abundant collagen of the human bodypresent in scar tissue, the end product when tissue heals by repair;Type II collagen—Auricular cartilage Type III collagen—This is thecollagen of granulation tissue, and is produced quickly by youngfibroblasts before the tougher type I collagen is synthesized; Type IVcollagen—Basal lamina; Type V collagen—most interstitial tissue, assoc.with type I; Type VI collagen—most interstitial tissue, assoc. with typeI; Type VII collagen—epithelia; Type VIII collagen—some endothelialcells; Type IX collagen—cartilage, assoc. with type II; Type Xcollagen—hypertrophic and mineralizing cartilage; Type XIcollagen—cartilage; Type XII collagen—interacts with types I and III.

Within the context of certain embodiments, “collagen modulatingsubstances” means a variety of substances capable of facilitating theformation or breaking down of units or of any type of collagen.

A “gel” is a semisolid material formed from a colloidal solution. Byweight, gels are mostly liquid, yet they behave like solids. An exampleis gelatin.

“Keratin” is any of a variety of fibrous protein molecules that serve asstructural units for various living tissues. The keratins are the majorprotein components of hair, wool, nails, horn, hoofs, and the quills offeathers. These proteins generally contain large quantities of thesulfur-containing amino acids, particularly cysteine. The helicalkeratin molecules twist around each other to form elongated strandscalled intermediate filaments. The formation of a disulfide bridgebetween the sulfur atoms on two cysteines on separate polypeptide chainsof keratin allows for the cross-linkage of these chains and results in afairly rigid aggregate.

“Filaggrin” is any of a variety of filament-associated proteins thatinteract with keratin intermediate filaments of terminallydifferentiating mammalian epidermis via disulphide bond formation.

“Immunomodulator” means any of a variety of substance that influencesthe immune system. Examples include, but are not limited to, cytokines,Interleukin-2, immunostimulants, and immunosuppressors.

The term “natural product” means any of a variety of organic chemicalmoieties whose molecular arrangement is derived from enzymatictransformations in a living organism excluding amino acids, proteins,polypeptides, nucleic acids and sequences, and saturated fatty acids.Examples include, but are not limited to lipids (i.e., that are notsaturated fatty acids), carbohydrates/saccharides and polysaccharides,the steroids and their derivatives, the terpenes and their derivatives,vitamins, carotenoids, and natural medicines such as taxol, etc. Theterm “synthetic natural product” is a natural product not obtained fromits natural source.

The term “gene” as used herein, refers to a DNA sequence that comprisescontrol and coding sequences necessary for the production of apolypeptide or protein precursor. The polypeptide can be encoded by afull length coding sequence or by any portion of the coding sequence, aslong as the desired protein activity is retained.

“Nucleoside,” as used herein, refers to a compound consisting of apurine [guanine (G) or adenine (A)] or pyrimidine [thymine (T), uridine(U), or cytidine (C)] base covalently linked to a pentose, whereas“nucleotide” refers to a nucleoside phosphorylated at one of its pentosehydroxyl groups.

“Nucleic acid sequence” as used herein refers to an oligonucleotide,nucleotide or polynucleotide, and fragments or portions thereof, and toDNA or RNA of genomic or synthetic origin that may be single- ordouble-stranded, and represent the sense or antisense strand.

An “amino acid sequence” as used herein refers to a peptide or proteinsequence.

A “peptide nucleic acid” as used herein refers to an oligomeric moleculein which nucleosides are joined by peptide, rather than phosphodiester,linkages. These small molecules, also designated anti-gene agents, stoptranscript elongation by binding to their complementary (template)strand of nucleic acid (Nielsen et al. (1993) Anticancer Drug Des.,8:53-63).

A “variant” in regard to amino acid sequences is used to indicate anamino acid sequence that differs by one or more amino acids fromanother, usually related amino acid. The variant may have “conservative”changes, wherein a substituted amino acid has similar structural orchemical properties (e.g., replacement of leucine with isoleucine). Morerarely, a variant may have “non-conservative” changes, e.g., replacementof a glycine with a tryptophan. Similar minor variations may alsoinclude amino acid deletions or insertions (i.e., additions), or both.Guidance in determining which and how many amino acid residues may besubstituted, inserted or deleted without abolishing biological orimmunological activity may be found using computer programs well knownin the art, for example, DNAStar software.

As used herein the term “portion” in reference to an amino acid sequenceor a protein (as in “a portion of an amino acid sequence”) refers tofragments of that protein. The fragments may range in size from fouramino acid residues to the entire amino acid sequence minus one aminoacid.

As used herein, the term “purified” refers to molecules, including butnot limited to nucleic, ribonucleic, lipid or amino acid sequences,which are removed from their natural environment, isolated or separated.An “isolated nucleic acid sequence” is therefore a purified nucleic acidsequence. “Substantially purified” molecules are at least 60% free,preferably at least 75% free, and more preferably at least 90% free fromother components with which they are naturally associated.

“Cancer” means any of various cellular diseases with malignant neoplasmscharacterized by the proliferation of anaplastic cells. It is notintended that the diseased cells must actually invade surrounding tissueand metastasize to new body sites. Cancer can involve any tissue of thebody and have many different forms in each body area. Most cancers arenamed for the type of cell or organ in which they start.

“Cell” means the smallest structural unit of living matter capable offunctioning autonomously, consisting of one or more nuclei, cytoplasm,and various organelles, all surrounded by a semipermeable membrane.Cells include all somatic cells obtained or derived from a living ordeceased animal body at any stage of development as well as germ cells,including sperm and eggs (animal reproductive body consisting of an ovumor embryo together with nutritive and protective envelopes). Includedare both general categories of cells: prokaryotes and eukaryotes. Thecells contemplated for use in this invention include all types of cellsfrom all organisms in all kingdoms: plans, animals, protists, fungi,archaebacteria and eubacteria. Stem cells are cells capable, bysuccessive divisions, of producing specialized cells on many differentlevels. For example, hematopoietic stem cells produce both red bloodcells and white blood cells. From conception until death, humans containstem cells, but in adults their power to differentiate is reduced.

As used herein, the term “differentiation” related to cells means theprocess by which cells becomes structurally and functionallyspecialized, which is a progressive restriction of the developmentalpotential and increasing specialization of function which takes placeduring the development of the embryo and leads to the formation ofspecialized cells, tissues, and organs.

The term “dedifferentiation” related to cells means the reverse processof differentiation, where cells become less structurally andfunctionally specialized, which increases the developmental potential ofthe cell.

“Differentiable” means the ability of a cell to differentiate into adesired cell type. As used herein, the term “differentiates” meansspecialization (differentiation) or return to a more primitive celltype; dedifferentiation).

An “extract” as used in the context of “cell extract” and “egg extract”in this invention means a preparation of any type of cell as definedabove obtained by chemical or mechanical action, as by pressure,distillation, evaporation etc. Extracts can include all or any singlecomponent or combination of components of the cells, includingconcentrated preparations of the active components. Such components ofthe extracts include but are not limited to RNA, DNA, lipids, all aminoacid base structures including peptides and proteins, carbohydrates orcombinations thereof. Extracts contemplated by this invention includebut are not limited to extracts of fish eggs, urchin eggs, frog eggs,adult stem cells, plant seeds and plant stem cells.

“Growth media” are compositions used to grow microorganisms or cells inculture. There are different sorts of media for growing different sortsof cells. The biggest difference in growth media are between those usedfor growing cells in culture (cell culture uses specific cell typesderived from plants or animals) and those used for growingmicroorganisms (usually bacteria or yeast). These differences arise dueto the fact that cells derived from whole organisms and grown in cultureare often incapable of growth without the provision of certainrequirements, such as hormones or growth factors which usually occur invivo. In the case of animal cells these requirements are often providedby the addition of blood serum to the medium. These media are often redor pink due to the inclusion of pH indicators. Growth media forembryonic stem cells preferably contains minimal essential medium, i.e.,Eagle's: amino acids, salts (Ferric nitrate nonahydrate, Potassiumchloride, Magnesium sulfate, Sodium chloride, Sodium dihydrogenphosphate), vitamins, (Ascorbic acid, Folic acid, Nicotinamide,Riboflavin, B-12) or Dulbecco's: additionally iron, glucose;non-essential amino acids, sodium pyruvate, β-mercaptoethanol,L-glutamine, fetal bovine serum and Leukemia Inhibitory Factor (LIF). Inthe case of microorganisms, there are no such limitations as they areoften single cell organisms. One other major difference is that animalcells in culture are often grown on a flat surface to which they attach,and the medium is provided in a liquid form, which covers the cells.Bacteria such as Escherichia coli (E. coli, the most commonly usedmicrobe in laboratories) may be grown on solid media or in liquid media,liquid nutrient medium is commonly called nutrient broth. The preferredgrowth media for microorganisms are nutrient broth or Luria-Bertanimedium (L-B medium). Bacteria grown in liquid cultures often formcolloidal suspensions. When agar (a substance which sets into a gel) isadded to a liquid medium it can be poured into Petri dishes where itwill solidify (these are called agar plates) and provide a solid mediumon which microbes may be cultured.

Within the context of certain embodiments, “to glue to skin” means tostick or fasten to with or as if with any of various adhesives, such as,glue, paste or mucilage.

A “lipid” means any of a group of organic compounds, including the fats,oils, waxes, sterols, and triglycerides that are insoluble in water butsoluble in nonpolar organic solvents, and are oily to the touch. Majorclasses of lipids include the fatty acids, the glycerol-derived lipids(including the fats and oils and the phospholipids), thesphingosine-derived lipids (including the ceramides, cerebrosides,gangliosides, and sphingomyelins), the steroids and their derivatives,the terpenes and their derivatives, certain aromatic compounds, andlong-chain alcohols and waxes. In living organisms lipids serve as thebasis of cell membranes and as a form of fuel storage. Often lipids arefound conjugated with proteins or carbohydrates, and the resultingsubstances are known as lipoproteins and lipopolysaccharides. Thefat-soluble vitamins can be classified as lipids. Liposomes arespherical vesicles formed by mixing lipids with water or watersolutions. They have found applications in the oral administration ofsome drugs (e.g., insulin and some cancer drugs), since they retaintheir integrity until they are broken down by the lipases in the stomachand small intestine.

Within the context of certain embodiment, a “nutrient gel layer” a gelcomprising substances typically contained in a growth medium.

Within the context of certain embodiments, “specialized cell” of asubject means that the cell has characteristic immunoidentificativemarkers, such that differentiation of these cells and exposure totissues of the subjects can be done under conditions such that immunesystem does not create antibodies to the differentiated cells. Forexample, when red blood cells carrying one or both A or B antigens areexposed to the corresponding antibodies, they agglutinate; that is,clump together. People usually have antibodies against those red cellantigens that they lack. Thus, specialized red blood cells of thesubject would be those of the proper blood type. The cause of transplantrejection is recognition of foreign MHC antigens by T cells andactivation of those T cells to become effector cytotoxic or helper Tcells. T cell activation occurs in the case of vascularized grafts ofnucleated cells expressing WIC Matching MHC Class I (especially HLA-B)and Class II HLA-DR alleles is more important for successfultransplantation than matching other WIC antigens; and matching WIC ismore important than matching minor histocompatibility antigens. Thus,specialized MHC presenting cells of the subject would be thosepresenting matching MHC alleles.

The term “manage” when used in connection with a disease or conditionmeans to provide beneficial effects to a subject being administered witha prophylactic or therapeutic agent, which does not result in a cure ofthe disease. In certain embodiments, a subject is administered with oneor more prophylactic or therapeutic agents to manage a disease so as toprevent the progression or worsening of the disease.

As used herein, the terms “prevent” and “preventing” include theprevention of the recurrence, spread or onset. It is not intended thatthe present invention be limited to complete prevention. In someembodiments, the onset is delayed, or the severity of the disease isreduced.

As used herein, the terms “treat” and “treating” are not limited to thecase where the subject (e.g. patient) is cured and the disease iseradicated. Rather, the present invention also contemplates treatmentthat merely reduces symptoms, and/or delays disease progression.

Within the context of certain embodiments, a “waterproof layer” means amaterial or fabric that is substantially impervious to water or a layerof a sealing agent to intended to prevent substantial penetration bywater.

As used herein, the term “transport vehicle” includes substances capableof aiding penetration of intact skin or skin cells or other somaticcells. The term “transport vehicle” is used synonymously with the term“permeabilizing agents”. Such transport vehicles include, but are notlimited to: phospholipids, palmitylmyristyrates, DMSO, polymer orchitosan suspensions or matrix, liposomes, Trojan peptides, chariotpeptides, small elastic vesicles, microspheres (functionalized vectorsmade from naturally derived materials such as collagen,glycosaminoglycans, chondroitin sulfate, chitosan or polysaccharides),nanoparticles (carries lipophilic substances and enhance bioavailabilityof the encapsulated material into skin), preloaded spherical beads andsponges, uni- and/or multilamellar vesicles (stabilize contents ofextracts in cream base and help transport into skin), retinol molecularfilm fluid (thin uniform monolayer film that facilitates the transfer ofactives through the stratum corneum), poly acrylo nitrile (polymerscomprising a controlled release system that synchronizes the release ofan active ingredient along with a fragrance as a sensory marker whichconveys the efficacy of the product), beta-glucan (oat fiber which aidsin penetration of the skin, (Redmond, Int. Journ. Cosmetic science2005), propylene glycol (as drug carrier, work best with a mineral oilbased cream/lotion etc), butylene glycol, polyethylene glycol, oliveoil, dimethyl isosorbide, dimethylformamide, methyl salicylate (theseall enhance absorption through skin), long chain oleic acids (disruptsthe bilayer within the stratum corneum, vital for permeation ofcompositions in propylene glycol-based formulations), substances capableof adjusting pH, hydration and local metabolism in skin. Agentsmodifying these factors include a vehicle containing an activehydrophobic agent, de-ionization of active ingredients, increasedhydration of the skin (water content of carrier solution/cream/medium),lactic acid (alters the pH).

As used herein, the term “NANOG” refers to a homeobox gene. NANOG isthought to be required for stem cells to multiply without limit whileremaining able to make many different types of cells. The gene is apotential master gene that helps make embryonic stem cells grow in thelaboratory, making stem cells immortal.

As used herein, the term “OCT4” refers to a gene that is not active insomatic cells, including adult stem cells, but is expressed in embryonicstem and germ cells. OCT4 is essential to maintain pluripotency ofembryonic stem cells.

As used herein, the term “SOX2” refers to the sex determining region Y(SRY) box 2 protein coding gene. This intronless gene encodes a memberof the SRY-related HMG-box (SOX) family of transcription factorsinvolved in the regulation of embryonic development and in thedetermination of cell fate.

As used herein, the term “GAPDH” refers to the housekeeping geneglyceraldehydes-3-phosphate dehydrogenase. This gene is involved inbasic functions needed for cell maintenance. Housekeeping genes areconstitutively expressed.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to improving health and damage of cells andtissues preferably skin, and more preferably restoring aged or damagedskin to a youthful and healthy appearance. In some embodiments, theinvention relates to compositions of cells, cell or egg extracts, andextract components which can induce differentiation, including but notlimited to purified or synthetic nucleic acid sequences, polypeptides,or natural products contained in said extracts. In some embodiments, thecells are differentiable cells, preferably stem cells or eggs. In somepreferred embodiments, the extracts are aqueous extracts. In someembodiments, the extracts are from a non-avian source. In someembodiments, compositions are used in a method that comprisesapplication of compositions to skin and/or wounds after removal theouter surface layers. In some embodiments, the invention related to amethod of dedifferentiation of cells and/or dedifferentiation followedby redifferentiation. In some embodiments, the invention relates tomanaging, preventing, and treating skin diseases.

Application of the composition to the desired surface may beprophylactic, so that the composition is applied to the skin or othersurface before exposure to an agent occurs. Application of thecomposition may be curative, for example, to further protect acompromised skin surface or to provide a protectant surface duringnatural or mediated healing of an exposed skin surface. Application ofthe composition may be protective, for example, to protect a skinsurface should exposure to the agent occur.

The present invention relates to the use of extracts or components ofdifferentiable cells for topical application to surfaces of the body.Accordingly, the present invention provides methods and compositions forcosmetic and therapeutic uses. The present invention is not limited tothe use of extracts or components of any particular type ofdifferentiatable cell. Indeed, the use of variety of types of cells anddifferentiable cells from any organism is contemplated, including, butnot limited to, mammalian embryonic stem cells, mammalian adult stemcells, cord blood cells, fish, shrimp or sea urchin eggs and embryos,and amphibian eggs and embryos.

In some embodiments, the invention relates to dedifferentiating existingepithelial/epidermal cells to a primordial state, wherein the cells havestem-cell capacities and can reform the correct and needed cells for theregeneration of the whole layer of skin (epidermis, dermis andsubdermis). Although many differentiated cells are typically committedto their fate, dedifferentiation events can take place. Urodeleamphibians and teleost fish can replace lost anatomical parts by aprocess of migration, dedifferentiation, proliferation andredifferentiation of epithelial cells in the wounded area. Functionalreprogramming of differentiated cell nuclei has also been illustrated bythe derivation of pluripotent embryonic stem cells (ESCs), and by thelive birth of cloned animals after nuclear transplantation intounfertilized eggs.

The term plasticity, as used in this herein, means that a cell from onetissue can generate the differentiated cell types of another tissue.Xenopus eggs can reprogram mammalian somatic nuclei to express the POUfamily member homeodomain transcription factor gene Oct4 by a processrequiring DNA demethylation. DNA demethylation also occurs after fusionof mouse thymocytes with embryonic germ cells (EGCs) but interestingly,only EG cells are capable of demethylating imprinted genes. Fusion ofneuronal progenitor cells or bone marrow derived cells with ESCs resultsin hybrids which express markers of pluripotency. Similar results areobtained from fusing human fibroblasts with ESCs. Fusion of embryonalcarcinoma cells (ESCs) with T-lymphoma cells also promotes the formationof colonies expressing pluripotent cell transcripts from the lymphomagenome. Components of pluripotent EG, ES or EC cells can elicitreprogramming events in a somatic genome.

Somatic nuclear function can be altered using nuclear and cytoplasmicextracts because extracts provide the necessary regulatory components.Extracts of regenerating newt limbs promote cell cycle reentry anddownregulation of myogenic markers in differentiated myotubes.Teratocarcinomas are a particular type of germ cell tumors which containundifferentiated stem cells and differentiated derivatives that caninclude endoderm, mesoderm and ectoderm germ layers. Undifferentiatedcarcinoma cells can be cultured to give rise to lines of ECCs. ECCs formmalignant teratocarcinomas when transplanted into ectopic sites;however, some ECC lines can also contribute to tissues of the developingfetus when introduced into a blastocyst.

Undifferentiated human teratocarcinoma NCCIT cells can be establishedfrom a mediastinal mixed germ cell tumor. NCCIT is at a stageintermediate between a seminoma (a precursor of germ cell tumors) and anembryonal carcinoma. NCCIT is a developmentally pluripotent cell linethat can differentiate into derivatives of all three embryonic germlayers and extraembryonic cell lineages an extract of undifferentiatedsomatic cells can elicit dedifferentiation in a somatic cell line. SeeTaranger et al., “Induction of Dedifferentiation, Genome-wideTranscriptional Programming, and Epigenetic Reprogramming by Extracts ofCarcinoma and Embryonic Stem Cells” Mol. Biol. Cell. (2005).

Stem cells can establish in damaged tissue. See Menard et al.,“Transplantation of cardiac-committed mouse embryonic stem cells toinfarcted sheep myocardium: a preclinical study” Lancet,366(9490):1005-12 (2005); Goldman “Stem and progenitor cell-basedtherapy of the human central nervous system” Nat Biotechnol.23(7):862-71 (2005); Leri et al., “Repair of the damaged heart” KidneyInt. 68(5):1962 (2005); Levy et al., “Embryonic and adult stem cells asa source for cell therapy in Parkinson's disease” J Mol Neurosci.24(3):353-86 (2004); Jack et al., “Processed lipoaspirate cells fortissue engineering of the lower urinary tract: implications for thetreatment of stress urinary incontinence and bladder reconstruction” JUrol. 174(5):2041-5 (2005); Kitmaura et al., Establishment of renalstem/progenitor-like cell line from S3 segment of proximal tubules inadult rat kidney, Kidney Int. 68(5):1966 (2005).

In some embodiments, the invention relates to extracts that are capableof stimulating the immune system to aid in healing. For example, theextracts may contain fibrogen and heat shock proteins. These endogenouscellular components are alarm signals typically expressed in distressedor injured cells. They bind Toll-like receptors (TLRs) in antigenpresenting cells (APCs) and put the immune system on alert of a damagedarea. See Matzinger “The Danger Model: A Renewed Sense of Self” Science296:301-305 (2002).

In some embodiments, the invention relates to stimulating existing stemcells in skin, such as stem cells found in and around hair follicles toduplicate and/or differentiate into epithelial cells or neurons. Nestin,a marker for neural progenitor cells, is expressed in cells of thehair-follicle bulge and behave as stem cells, differentiating to formmuch of the hair follicle during each hair growth cycle. The hairfollicle is dynamic, cycling between growth (anagen), regression(catagen), and resting (telogen) phases throughout life. Stem cellslocated in the hair-follicle bulge area give rise to the folliclestructures during each anagen phase. Bulge hair-follicle stem cells cangenerate all epithelial cell types within the intact follicle and hairduring normal hair-follicle cycling. The bulge hair-follicle stem cellsdifferentiate into hair-follicle matrix cells, sebaceous-gland basalcells, and epidermis. In response to wounding, some stem cells exit thebulge, migrate, and proliferate to repopulate the infundibulum andepidermis. Multipotent adult stem cells from the skin dermis, termedskin-derived precursors (SKPs), can proliferate and differentiate toproduce neurons, glia, smooth muscle cells, and adipocytes. Pluripotentneural crest stem cells are present in the dermal papillae of adultmammalian hair follicles. See Amoh et al., “Multipotent nestin-positive,keratin-negative hair-follicle bulge stem cells can form neurons” Proc.Natl. Acad. Sci. USA. 12; 102(15):5530-4 (2005).

The bone marrow contains three stem cell populations—hematopoietic stemcells, stromal cells, and endothelial progenitor cells. Bone marrow stemcells, the hematopoietic stem cells (HSCs), are responsible for formingall of the types of blood cells in the body. The bone marrow-derivedcells are sometimes sorted—using a panel of surface markers—intopopulations of hematopoietic stem cells or bone marrow stromal cells.The HSCs may be highly purified or partially purified, depending on theconditions used. Another way to separate population of bone marrow cellsis by fractionation to yield cells that adhere to a growth substrate(stromal cells) or do not adhere (hematopoietic cells). The mesenchymalstem cells of the bone marrow also give rise to these tissues, andconstitute the same population of cells as the bone marrow stromalcells. Progenitor cells that differentiates into endothelial cells, atype of cell that lines the blood vessels, can be isolated fromcirculating blood. Pericytes are related to bone marrow stromal cells.

Combinations of surface markers are used to identify, isolate, andpurify HSCs derived from bone marrow and blood. Undifferentiated HSCsand hematopoietic progenitor cells express c-kit, CD34, and H-2K. Thesecells usually lack the lineage marker Lin, or express it at very lowlevels (Lin−/low). BM stromal cells have several features thatdistinguish them from HSCs. The two cell types are separable in vitro.When bone marrow is dissociated, the mixture of cells it contains isplated at low density, the stromal cells adhere to the surface of theculture dish, and the HSCs do not. Given specific in vitro conditions,BM stromal cells form colonies from a single cell called the colonyforming unit-F (CFU-F). These colonies may then differentiate asadipocytes or myelo supportive stroma, a clonal assay that indicates thestem cell-like nature of stromal cells. Unlike HSCs, which do not dividein vitro (or proliferate only to a limited extent), BM stromal cells canproliferate for up to 35 population doublings in vitro. Endothelial stemcells are CD34+(a marker for HSCs), and they express the transcriptionfactor GATA-2 see Kocher, et al., “Neovascularization of ischemicmyocardium by human bone-marrow-derived angioblasts preventscardiomyocyte apoptosis, reduces remodeling and improves cardiacfunction,” Nat. Med. 7, 430-436 (2001).

The present invention contemplates the use of any type of cell includingstem cells from any multicellular organism in any kingdom of species,both eukaryotes including animals, plants, protists, fungi, andprokaryotes including the kingdoms archaebacteria and eubacteria.Multicellular organisms contain totipotent, mulitpotent, pluripotent andunipotent stem cells capable of dividing and replenishing tissues andcells which compose the organism. Stem cells are well documented inmammalian animals, but are present in all animals, e.g. insects. Adultfruit flies have the same stem cells controlling cell regulation intheir gut as humans do. Vertebrate and invertebrate digestive systemsshow extensive similarities in their development, cellular makeup andgenetic control. The Drosophila midgut is typical: enterocytes make upthe majority of the intestinal epithelial monolayer, but areinterspersed with hormone-producing enteroendocrine cells. Human (andmouse) intestinal cells are continuously replenished by stem cells, themisregulation of which may underlie some common digestive diseases andcancer. In contrast, stem cells have not been described in theintestines of flies, and Drosophila intestinal cells have been thoughtto be relatively stable. By lineage labeling it has been shown thatadult Drosophila posterior midgut cells are continuously replenished bya distinctive population of intestinal stem cells (ISCs). (BenjaminOhlstein and Allan Spradling, The adult Drosophila posterior midgut ismaintained by pluripotent stem cells, Nature Online Dec. 7, 2005).

In addition to animal stem cells, plants also contain stem cells. Stemcells in plant shoot and root meristems are maintained throughout thelife of the plant and produce somatic daughter cells that make up thebody of the plant. Plant stem cells can also be derived from somaticcells in vivo and in vitro. (Plants stem cells: divergent pathways andcommon themes in shoots and roots. Byrne M E, Kidner C A, Martienssen RA. Curr Opin Genet Dev. 2003 October; 13(5):551-7.) Animal cells andorganisms move, conduct cell divisions which serve to regenerate andmaintain tissues and circulating cell populations, grow in aconcurrently repetitive manner, contain a reserved germline set aside inembryogeny, have a low tolerance to genetic abnormalities, produceembryos complex and incomplete, and display essentially no asexualpropagation and have no cell wall. Plants respond by physiologicaladjustment, their cell divisions contribute to de novo formation oforgans all the way through to senescence, plant growth is serial,repetitive, and plastic, plants have no reserved germline, are moretolerant of genetic abnormalities, their embryos simple and complete,and plant cells are totipotent. Plant stem cells and seeds (plantgametes) are contemplated for use in this invention. Contrary to therarity of totipotent cells in animals, almost every cell formed by afungus can function as a “stem cell”. The multicellular fruiting bodiesof basidiomycete fungi consist of the same kind of filamentous hyphaethat form the feeding phase, or mycelium, of the organism, and visiblecellular differentiation is almost nonexistent (Money N P. Mushroom stemcells. Bioessays. 2002 October; 24(10):949-52).

The description is organized into the following sections: A. Mammalianembryonic stem cell extracts; B. Adult stem cell extracts; C. Cord bloodcell extracts; D. Non-mammalian cell, egg and embryo extracts; E.Methods for preparing extracts; F. Epigenetic inhibitors; G. Topicaldelivery methods; H. Other delivery methods; I. Additional componentsfor extracts; J. Composition profiles; K. Topical application; L.Therapeutic uses; M. Whole cell applications; N. Ex vivo and in vivotherapy.

A. Mammalian Embryonic Stem Cell Extracts

In some embodiments, the present invention provides compositionscomprising embryonic stem cells or extracts prepared from embryonic stemcells. In some preferred embodiments, the cells or extracts areformulated for topical application as described in more detail below.The present invention is not limited to the use of any particular typeof embryonic stem cells. Indeed, the use of embryonic stem cells from anumber of animal species is contemplated, including all species in theanimal kingdom, but not limited to invertebrates and vertebrates,including species in the phylum chordata, including all classes, andimportantly all orders of the class mammalia, including but not limitedto all primates, rodents, carnivores, lagomorphs and artiodactyles.Methods for obtaining pluripotent cells from species in these animalorders, including monkeys, mice, rats, pigs, cattle and sheep have beenpreviously described. See, e.g., U.S. Pat. Nos. 5,453,357; 5,523,226;5,589,376; 5,340,740; and 5,166,065 (all of which are specificallyincorporated herein by reference); as well as, Evans, et al.,Theriogenology 33(1):125-128, 1990; Evans, et al., Theriogenology33(1):125-128, 1990; Notarianni, et al., J. Reprod. Fertil.41(Suppl.):51-56, 1990; Giles, et al., Mol. Reprod. Dev. 36:130-138,1993; Graves, et al., Mol. Reprod. Dev. 36:424-433, 1993; Sukoyan, etal., Mol. Reprod. Dev. 33:418-431, 1992; Sukoyan, et al., Mol. Reprod.Dev. 36:148-158, 1993; Iannaccone, et al., Dev. Biol. 163:288-292, 1994;Evans & Kaufman, Nature 292:154-156, 1981; Martin, Proc Natl Acad SciUSA 78:7634-7638, 1981; Doetschman et al. Dev Biol 127:224-227, 1988);Giles et al. Mol Reprod Dev 36:130-138, 1993; Graves & Moreadith, MolReprod Dev 36:424-433, 1993 and Bradley, et al., Nature 309:255-256,1984.

Primate embryonic stem cells may be preferably obtained by the methodsdisclosed in U.S. Pat. Nos. 5,843,780 and 6,200,806, each of which isincorporated herein by reference. Primate (including human) stem cellsmay also be obtained from commercial sources such as WiCell, Madison,Wis. A preferable medium for isolation of embryonic stem cells is “ESmedium.” ES medium consists of 80% Dulbecco's modified Eagle's medium(DMEM; no pyruvate, high glucose formulation, Gibco BRL), with 20% fetalbovine serum (FBS; Hyclone), 0.1 mM β-mercaptoethanol (Sigma), 1%non-essential amino acid stock (Gibco BRL). Preferably, fetal bovineserum batches are compared by testing clonal plating efficiency of a lowpassage mouse ES cell line (ES_(jt3)), a cell line developed just forthe purpose of this test. FBS batches must be compared because it hasbeen found that batches vary dramatically in their ability to supportembryonic cell growth, but any other method of assaying the competenceof FBS batches for support of embryonic cells will work as analternative.

Primate ES cells are isolated on a confluent layer of murine embryonicfibroblast in the presence of ES cell medium. Embryonic fibroblasts arepreferably obtained from 12 day old fetuses from outbred CF1 mice(SASCO), but other strains may be used as an alternative. Tissue culturedishes are preferably treated with 0.1% gelatin (type I; Sigma).Recovery of rhesus monkey embryos has been demonstrated, with recoveryof an average 0.4 to 0.6 viable embryos per rhesus monkey per month,Seshagiri et al. Am J Primatol 29:81-91, 1993. Embryo collection frommarmoset monkey is also well documented (Thomson et al. “Non-surgicaluterine stage preimplantation embryo collection from the commonmarmoset,” J Med Primatol, 23:333-336 (1994)). Here, the zona pellucidais removed from blastocysts by brief exposure to pronase (Sigma). Forimmunosurgery, blastocysts are exposed to a 1:50 dilution of rabbitanti-marmoset spleen cell antiserum (for marmoset blastocysts) or a 1:50dilution of rabbit anti-rhesus monkey (for rhesus monkey blastocysts) inDMEM for 30 minutes, then washed for 5 minutes three times in DMEM, thenexposed to a 1:5 dilution of Guinea pig complement (Gibco) for 3minutes.

After two further washes in DMEM, lysed trophectoderm cells are removedfrom the intact inner cell mass (ICM) by gentle pipetting, and the ICMplated on mouse inactivated (3000 rads gamma irradiation) embryonicfibroblasts. After 7-21 days, ICM-derived masses are removed fromendoderm outgrowths with a micropipette with direct observation under astereo microscope, exposed to 0.05% Trypsin-EDTA (Gibco) supplementedwith 1% chicken serum for 3-5 minutes and gently dissociated by gentlepipetting through a flame polished micropipette.

Dissociated cells are replated on embryonic feeder layers in fresh ESmedium, and observed for colony formation. Colonies demonstratingES-like morphology are individually selected, and split again asdescribed above. The ES-like morphology is defined as compact colonieshaving a high nucleus to cytoplasm ratio and prominent nucleoli.Resulting ES cells are then routinely split by brief trypsinization orexposure to Dulbecco's Phosphate Buffered Saline (without calcium ormagnesium and with 2 mM EDTA) every 1-2 weeks as the cultures becomedense. Early passage cells are also frozen and stored in liquidnitrogen.

In some embodiments, extracts are prepared from the mammalian embryonicstem cells. In some embodiments, cells are washed in phosphate bufferedsaline (PBS) and in cell lysis buffer (100 mM HEPES, pH 8.2, 50 mM NaCl,5 mM MgCl₂, 1 mM dithiothreitol and protease inhibitors), sedimented at400 g, resuspended in 1 volume of cold cell lysis buffer and incubatedfor 30-45 min on ice to allow swelling. Cells are sonicated on ice in200-μ1 aliquots using a Labsonic-M pulse sonicator fitted with a 3-mmdiameter probe (B. Braun Biotech, Melsungen, Germany) until all cellsand nuclei are lysed. The lysate is sedimented at 15,000 g for 15 min at4° C. to pellet the coarse material. The supernatant is aliquoted,frozen in liquid nitrogen and can be stored for up to 9 months at −80°C. If necessary, extracts can be diluted with H₂O prior to use to adjustthe osmolarity to ˜300 mOsm (i.e., isotonicity).

In some embodiments, the animal stem cell extracts, including but notlimited to mammalian stem cell extracts, are used as is, while in otherembodiments, the extracts are formulated either alone or with othercomponents as described in more detail below.

B. Adult Stem Cell Extracts

In some embodiments, the present invention provides compositionscomprising adult stem cells or extracts prepared from adult stem cells.In some preferred embodiments, the cells or extracts are formulated fortopical application as described in more detail below. The adult stemcell is an undifferentiated (unspecialized) cell that is found in adifferentiated (specialized) tissue; it can renew itself and becomespecialized to yield specialized cell types of the tissue from which itoriginated. These precursor cells exist within the differentiatedtissues of the adult of all multicellular organisms in the animal,plant, protist and fungi kingdoms as a community of cells dispersedthroughout the tissue. Precursor cells derived from adults can bedivided into three categories based on their potential fordifferentiation. These three categories of precursor cells areepiblast-like stem cells, germ layer lineage stem cells, and progenitorcells. Precursor cells have been isolated from a wide variety oftissues, including, but not limited to, skeletal muscle, dermis, fat,cardiac muscle, granulation tissue, periosteum, perichondrium, brain,meninges, nerve sheaths, ligaments, tendons, blood vessels, bone marrow,trachea, lungs, esophagus, stomach, liver, intestines, spleen, pancreas,kidney, urinary bladder, and testis. Precursor cells can be releasedfrom the connective tissue compartments throughout the body bymechanical disruption and/or enzymatic digestion and have been isolatedfrom, but not limited to, newborns, adolescent, and geriatric mice, ratsand humans, and adult rabbits, dogs, goats, sheep, and pigs.

The first category of precursor cells, epiblast-like stem cells (ELSCs),consists of a stem cell that will form cells from all three embryonicgerm layer lineages. Stem cells from adult rats and stem cells fromadult humans can be released from the connective tissue compartmentsthroughout the body by mechanical disruption and/or enzymatic digestion.The stem cells from either adult rats or adult humans can bepreferentially slow frozen and stored at −80° C.±5° C. using 7.5%ultra-pure dimethyl sulfoxide. Fast thawing of stem cells from bothspecies from the frozen state to ambient temperature yields recoveryrates exceeding 98%. These cells in the undifferentiated state expressthe Oct-3/4 gene that is characteristic of embryonic stem cells. ELSCsdo not spontaneously differentiate in a serum free environment lackingprogression agents, proliferation agents, lineage-induction agents,and/or inhibitory factors, such as recombinant human leukemia inhibitoryfactor (LIF), recombinant murine leukemia inhibitory factor (ESGRO), orrecombinant human anti-differentiation factor (ADF). Embryonic stemcells spontaneously differentiate under these conditions. In contrast,ELSCs derived from both species remain quiescent unless acted upon byspecific proliferative and/or inductive agents and/or environment.

ELSCs proliferate to form multiple confluent layers of cells in vitro inthe presence of proliferation agents such as platelet-derived growthfactors and respond to lineage-induction agents. ELSCs respond tohepatocyte growth factor by forming cells belonging to the endodermallineage. Cell lines have expressed phenotypic markers for many discretecell types of ectodermal, mesodermal, and endodermal origin when exposedto general and specific induction agents.

The second category of precursor cells consists of three separate stemcells. Each of the cells forms cells of a specific embryonic germ layerlineage (ectodermal stem cells, mesodermal stem cells and endodermalstem cells). When exposed to general and specific inductive agents, germlayer lineage ectodermal stem cells can differentiated into, forexample, neuronal progenitor cells, neurons, ganglia, oligodendrocytes,astrocytes, synaptic vesicles, radial glial cells, and keratinocytes.

The third category of precursor cells present in adult tissues iscomposed of a multitude of multipotent, tripotent, bipotent, andunipotent progenitor cells. In solid tissues these cells are locatednear their respective differentiated cell types. Progenitor cells do nottypically display phenotypic expression markers for pluripotent ELSCs,such as stage specific embryonic antigen-4, stage-specific embryonicantigen-1 or stage-specific embryonic antigen-3, or carcinoembryonicantigen cell adhesion molecule-1. Similarly, progenitor cells do nottypically display phenotypic expression markers for germ layer lineagestem cells, such as nestin for cells of the ectodermal lineage orfetoprotein for cells of the endodermal lineage.

A progenitor cell may be multipotent, having the ability to formmultiple cell types. A precursor cell of ectodermal origin residing inthe adenohypophysisand designated the adenohypophyseal progenitor cellis an example of a multipotent progenitor cell. This cell will formgonadotrophs, somatotrophs, thyrotrophs, corticotrophs, and mammotrophs.Progenitor cells for particular cell lineages have unique profiles ofcell surface cluster of differentiation (CD) markers and unique profilesof phenotypic differentiation expression markers. Progenitor cells donot typically spontaneously differentiate in serum-free defined mediumin the absence of a differentiation agent, such as LIF or ADF. Thus,unlike embryonic stem cells which spontaneously differentiate underthese conditions, progenitor cells remain quiescent unless acted upon byproliferative agents (such as platelet-derived growth factor) and/orprogressive agents (such as insulin, insulin-like growth factor-I orinsulin-like growth factor-II).

Progenitor cells can regulate their behavior according to changingdemands such that after transplantation they activate from quiescence toproliferate and generate both new satellite cells and substantialamounts of new differentiated cells. For example, the contractile unitsof muscle are myofibers, elongated syncytial cells each containing manyhundreds of postmitotic myonuclei. Satellite cells are resident beneaththe basal lamina of myofibers and function as myogenic precursors duringmuscle regeneration. In response to muscle injury, satellite cells areactivated, proliferate, and differentiate, during which they fusetogether to repair or replace damaged myofibers. When satellite cellsare removed from their myofibers by a non-enzymatic physical titrationmethod, they retain their ability to generate substantial quantities ofnew muscle after grafting that they are not able to attain by enzymaticdigestion. Conventional enzymatic disaggregation techniques impairmyogenic potential. Collins and Partridge “Self-Renewal of the AdultSkeletal Muscle Satellite Cell” Cell Cycle 4:10, 1338-1341 (2005).

Accordingly, the present invention also contemplates the use ofnon-embryonic stem cells, such as those described above. In someembodiments, mesenchymal stem cells (MSCs) can be derived from marrow,periosteum, dermis and other tissues of mesodermal origin (See, e.g.,U.S. Pat. Nos. 5,591,625 and 5,486,359, each of which is incorporatedherein by reference). MSCs are the formative pluripotential blast cellsthat differentiate into the specific types of connective tissues (i.e.the tissues of the body that support the specialized elements;particularly adipose, areolar, osseous, cartilaginous, elastic, marrowstroma, muscle, and fibrous connective tissues) depending upon variousin vivo or in vitro environmental influences. Although these cells arenormally present at very low frequencies in bone marrow, various methodshave been described for isolating, purifying, and greatly replicatingthe marrow-derived mesenchymal stems cells in culture, i.e. in vitro(See also U.S. Pat. Nos. 5,197,985 and 5,226,914 and PCT Publication No.WO 92/22584, each of which are incorporated herein by reference).

Various methods have also been described for the isolation ofhematopoietic stem cells (See, e.g., U.S. Pat. Nos. 5,061,620;5,750,397; 5,716,827 all of which are incorporated herein by reference).It is contemplated that the methods of the present invention can be usedto produce lymphoid, myeloid and erythroid cells from hematopoietic stemcells. The lymphoid lineage, comprising B-cells and T-cells, providesfor the production of antibodies, regulation of the cellular immunesystem, detection of foreign agents in the blood, detection of cellsforeign to the host, and the like. The myeloid lineage, which includesmonocytes, granulocytes, megakaryocytes as well as other cells, monitorsfor the presence of foreign bodies in the blood stream, providesprotection against neoplastic cells, scavenges foreign materials in theblood stream, produces platelets, and the like. The erythroid lineageprovides the red blood cells, which act as oxygen carriers.

Accordingly, the present invention also contemplates the use of neuralstem cells, which are generally isolated from developing fetuses. Theisolation, culture, and use of neural stem cells are described in U.S.Pat. Nos. 5,654,183; 5,672,499; 5,750,376; 5,849,553; and 5,968,829, allof which are incorporated herein by reference. It is contemplated thatthe methods of the present invention can use neural stem cells toproduce neurons, glia, melanocytes, cartilage and connective tissue ofthe head and neck, stroma of various secretory glands and cells in theoutflow tract of the heart.

In some embodiments, extracts are prepared from the mammalian embryonicstem cells. In some embodiments, cells are washed in phosphate bufferedsaline (PBS) and in cell lysis buffer (100 mM HEPES, pH 8.2, 50 mM NaCl,5 mM MgCl₂, 1 mM dithiothreitol and protease inhibitors), sedimented at400 g, resuspended in 1 volume of cold cell lysis buffer and incubatedfor 30-45 min on ice to allow swelling. Cells are sonicated on ice in200-μ1 aliquots using a Labsonic-M pulse sonicator fitted with a 3-mmdiameter probe (B. Braun Biotech, Melsungen, Germany) until all cellsand nuclei are lysed. The lysate is sedimented at 15,000 g for 15 min at4° C. to pellet the coarse material. The supernatant is aliquoted,frozen in liquid nitrogen and can be stored for up to 9 months at −80°C. If necessary, extracts can be diluted with H₂O prior to use to adjustthe osmolarity to ˜300 mOsm (i.e., isotonicity).

In some embodiments, the adult stem cell extracts are used as is, whilein other embodiments, the extracts are formulated either alone or withother components as described in more detail below.

C. Cord Blood Cell Extracts

In some embodiments, the present invention provides compositionscomprising cord blood cells or extracts prepared from cord blood cells.In some preferred embodiments, the cells or extracts are formulated fortopical application as described in more detail below. Transplantationof umbilical-cord blood has been successfully performed to treatindividuals with blood-diseases; donors, used have been newborn siblingsbeing perfect HLA matches for the affects sibling. The advantages ofcord blood as a source of hematopoietic stem cells for transplantationare clear. First, the proliferative capacity of hematopoietic stem cellsin cord blood is superior to that of cells in marrow or blood fromadults. Because they proliferate rapidly, the stem cells in a singleunit of cord blood can reconstitute the entire hematopoietic system.Second, the use of cord blood reduces the risk of graft-versus-hostdisease, the main obstacle to the success of allogeneic transplantationof hematopoietic stem cells. Graft-versus-host disease is caused by areaction of T cells in the graft to HLA antigens in the recipient; theimmaturity of lymphocytes in cord blood dampens that reaction. A jointEuropean study showed that recipients of cord blood from HLA-identicalsiblings had a lower risk of acute or chronic graft-versus-host diseasethan recipients of marrow from HLA-identical siblings. Children withacute leukemia who received HLA-mismatched cord blood from an unrelateddonor also had a lower risk of graft-versus-host disease than recipientsof HLA-mismatched marrow from an unrelated donor (Hematopoieticstem-cell transplants using umbilical-cord blood, New England Journal ofMedicine, 2001, 344(24):1860-1861, editorial)

Cord blood cells from siblings or children with matching HLA could beused to make extracts or be applied for the use as contemplated by thisinvention.

D. Non-mammalian Cell, Egg and Embryo Extracts

In some embodiments, the compositions of the present invention utilizecell, egg and embryo extracts from vertebrates, including but notlimited to Superclass Gnathostomata (jawed vertebrates), Euteleostomi(bony vertebrates), Class Actinopterygii (ray-finned fishes), ClassSarcopterygii (lobe-finned fishes and terrestrial vertebrates),Tetrapoda (tetrapods), Amniota (amniotes), Synapsida (synapsids), ClassMammalia (mammals), Early Therapsida (early therapsids), Class Reptilia(reptiles), Anapsida (tortoises and turtles), Order Testudines(tortoises and turtles), Diapsida (birds, crocodiles, lizards, snakes,and relatives), Archosauria (birds and crocodiles), Order Crocodilia(caimans, crocodiles, and relatives), Lepidosauria (amphisbaenians,lizards, snakes, and tuataras), Order Rhynchocephalia (tuataras), OrderSquamata (amphisbaenians, lizards, and snakes), Class Amphibia(amphibians), Subclass Dipnoi (lungfishes), Actinistia, OrderCoelacanthiformes (coelacanths), Class Chondrichthyes (rays, sharks, andrelatives), Placodermi (armored fishes and placoderms), ClassCephalaspidomorphi, more preferably fish, shrimp, sea urchin oramphibian eggs or embryos. In some embodiments, unfertilized butactivated fish, shrimp, sea urchin or amphibian eggs are used. Thepresent invention is not limited to the use of any particular types ofeggs. Indeed, the use of a variety of eggs is contemplated, including,but not limited to eggs from Xenopus, shrimp, sea urchin, salmon, troutor zebrafish. In some embodiments, eggs are collected from maturefemales and spontaneously activate upon contact with water. In furtherembodiments, the eggs are washed in Ringer's saline. In someembodiments, the eggs are not from an avian species.

E. Preparation and Stabilization of Extracts and Fractions

Extracts of the present invention are prepared from any of the sourcesdescribed in section A-D. In some embodiments, the extracts are cellularextracts. Cellular extracts of the present invention are compositions ofdisrupted cells such as stem cells or eggs. The cells may be disruptedby a variety of methods, including, but not limited to, mechanicalshearing or blending, sonication, or osmotic lysis. In some embodiments,the cellular extracts are preferably further processed to yield acomposition that is substantially free of lipids naturally associatedwith the cells, such as cell membrane components. By substantially freeof lipids, it is meant that the cellular extract comprises less thanabout 1%, preferably less than about 0.5%, and more preferably less thanabout 0.1% of lipids that are naturally associated with the cells usedto make the cellular extract. In some embodiments, the extracts compriseless than about 1% and preferably less than 0.1% cholesterol orovalbumin. Accordingly, in some embodiments, the cellular extractcomprises carbohydrates, proteins, glycosylated or otherwise modifiedproteins, peptides, amino acids, RNA (mRNA, sRNA, miRNA, rRNA), DNA,water etc, and combinations thereof. In some embodiments, the cellularextracts can comprise small amounts of lipids naturally associated withthe cells, as well as nuclear components such as chromosomes, nucleicacids, and nuclear proteins. In some embodiments, the cellular extractis preferably a cytoplasmic extract or fraction prepared by removingnuclear, cell membrane and other water insoluble materials naturallyassociated with the cells. In some embodiments, these components areremoved by centrifugation or fractionation of the disrupted cells. Insome embodiments, the cellular extract is preferably an aqueous extractor fraction comprising water soluble cellular components such asproteins, mRNA, and carbohydrates.

A variety of methods may be used to prepare extracts. For example, insome embodiments, eggs are placed “dry” in a glass 15 ml centrifugetube, and crushed by sedimentation at 15,000 g for 15 min. This producesthree layers: a lipid top fraction, which is collected, aliquoted andfrozen; a middle cellular or cytoplasmic fraction, which is alsocollected, aliquoted and frozen; and a pellet fraction, which isdiscarded. In some embodiments, the cellular fraction or extractprimarily comprises contents of the cytoplasm. The cellular fraction isused as extract. In some embodiments, the cellular fraction may be usedin combination with a lipid fraction. The cytoplasmic fraction may becleared further by sedimentation at 50,000, 100,000 or 200,000 g toyield a further cellular extract which is primarily a water solubleextract fraction. Regardless of the fraction used, the extract can bediluted to about 300 mOsm with cell lysis buffer (see above), ifnecessary. Accordingly, in some preferred embodiments, a water solubleextract prepared from eggs or embryos is utilized.

In other embodiments, the eggs are suspended in 0.5 volume of cell lysisbuffer and sonicated on ice until all eggs are lysed. The particulatematerial is sedimented at 15,000 g for 15 min at 4° C. The supernatantconstitutes the extract. As above, osmolarity can be adjusted to 300mOsm if needed. The extract can also be cleared as above.

In still other embodiments, the eggs are suspended in cell lysis bufferas previously described. Eggs are lysed by Dounce homogenization using aglass mortar and pestle (Kontes, type A or B). The lysate is sedimentedand treated as described above.

In some preferred embodiments, the present invention providescompositions, either prepared from natural sources as described above orfrom artificial source materials, or a combination thereof. In someembodiments, the extracts are characterized as having an osmolarity offrom about 330 to 440, preferably about 350 mOsm. In some embodiments,the extracts have a pH of from about 5.0 to about 7.7, preferably a pHof about 6.5-7.0. In some embodiments, the extracts have a proteincontent of about 100 to 250 mg/ml, preferably about 160 to 190 mg/ml,and most preferably about 120 mg/ml. In some embodiments, thecompositions have a water content of about 20 to 90 percent waterweight/weight (w/w), preferably about 37 to 79% water w/w. In someembodiments, the extracts have a density of about 0.8 to about 1.4 g/ml,preferably about 1.1 g/ml. In some embodiments, the compositionscomprise trace elements including, but not limited to, calcium,phosphorus, zinc, copper and iron. In some embodiments, the compositionscomprise vitamins, including, but not limited to vitamins A, E,riboflavin, niacin, B 6, calcium pantothenate and B 12. In someembodiments, the present invention provides a fresh roe compositioncomprising: 2.7 to 3.4% protein; 3 to 5% carbohydrates; 1.0 to 1.7% fatsin the form of phospholipids, and 0.01 to 0.05% minerals in fresh roe,should be less fats and higher total protein in the extract), 37 to 79weight percent water. In some embodiments, the extracts further comprisea lipid fraction. In some embodiments, the lipid fraction comprises fromabout 60% to about 80% unsaturated fatty acids. In further embodiments,the compositions comprise phospholipids, including phosphatidyl cholines(lecithins) or as phosphatidyl ethanolamine (cephalins), and to a lesserextent inositol phosphatides, cerebrosides and sphingomyelines. In someembodiments, the lipid fraction is from about 0.1% to about 1%, 2%, 3%,4% or 5% of the total composition, while in other embodiments, thecompositions are substantially free or free of lipids.

In some embodiments, the artificial extracts are supplemented with 1)water, 2) any type of protein (BSA, albumin, vitellogenin, amino acidmixtures, etc.), 3) vitamins and minerals as described above, 4) saltsor osmoles to create osmolarity of approx 350 mOsm, 5) glycerol or otheragent to increase viscosity, 6) lipids such as lecithins, cephalins andother phospholipids, 7) carbohydrates, 8) growth factors such as FGF,EGF and IGF, 9) and chemo-attractants such as SLC/6Ckine/Exodus2/TCA4and CKbeta-11/MIP-3beta/ELC, 10) acid or base to adjust pH to 6.2-7.2,and 11) preservatives such as methyl paraben, propyl paraben, BHA orBHT.

In some embodiments, the eggs or extracts are treated to preventbacterial growth. The use of a variety of methods is contemplated. Insome embodiments, the following methods are combined. In someembodiments, unfertilized or fertilized eggs (e.g., fish or amphibianeggs) are treated prior to homogenization with a bactericidal orbacteristatic agent. Preferred agents include, but are not limited to,iodine containing agents such as betadine, buffodine, andpovidone-iodine, and other agents such as novasan, sodium hypochlorite,bacitracin, polymyxin B sulfate, silver containing compounds such assilver sulfadiazine and silver nitrate, mafenide acetate, nystatin,gentamicin, neomycin. In other embodiments, the extracts are treatedpost-homogenization to prevent bacterial growth. In some embodiments,the extracts, such as the cellular extracts or cytoplasmic fractions,are treated by heating. In some embodiments, the extracts are heated toabout 37, 40, 50, 60, 70, 80 or 90 degrees Celsius for about 30 secondsor 1, 2, 5, 10, 20, 30, 60 or 120 minutes.

In some embodiments, the eggs or extracts are filtered, preferablythrough 0.22 or 0.45 μm filters to remove bacteria. In some embodiments,before or after filtering, the extracts are treated by additionalcentrifugation (15 min-2 hrs) after heating the extract to 56° C. tospin down any bacteria present.

In other embodiments, eggs are washed in a sulfur-containing agent(e.g., calcium polysulphide or calcium thiosulphate (lime sulfur)) priorto preparation. In some embodiments, sulfur is added to the extracts toremove bacteria. In other embodiments, benzoyl peroxide is added to theextracts. In some embodiments, eggs are washed in 0.001% to about 0.2%by weight of a metal chlorite and sufficient acid to adjust the pH ofthe solution from about 2.2 to about 4.5 to remove bacteria. In furtherembodiments, the eggs and/or extract are placed in a vacuum drum andmixed with a natural solution containing salt, vitamin C or citric acid,and water to remove bacteria. In some embodiments, the eggs and/orextract are stirred, vortexed, sonicated, agitated or shaken with saltwater or liquid buffer to dislodge bacteria and vacuum filter off theliquid to remove bacteria. It will be possible to check bacterialcontent in the liquid and on the treated eggs for quality control. Insome embodiments, electrophoresis of the eggs and/or extract is used toremove bacteria. It is contemplated that such methods utilize theinfluences of electrical double layer, intensity of electrical field,electric density gradient, pH of the buffer solution, ionic strength ofbuffer solution, stage of growth of bacteria, and anion surface-activeagent upon the electrophoretic mobility of some species of bacteria.

In some embodiments, lipids are removed by treatments the homogenateprior to centrifugation or the extract after centrifugation. The use ofa variety of methods is contemplated. In some embodiments, lipids areremoved by filtering through fat-absorbing paper or filter by applying avacuum suction system to a container with a filter in the bottom, wherethe extract is placed in the container and suctioned through the filter.In some embodiments, lipids are removed by using an absorbent materialand an outer containment vessel. The extract is entered to a containerfilled with absorbent material through a pump and then recovered byapplying a vacuum. In some embodiments, lipids are removed with hollowfiber contraction systems and/or extraction solvents for removing lipidsfrom viscous fluids, where contact a fluid with an extraction solvent,which causes the lipids in the fluid to separate from the fluid orcauses lipids in the lipid-containing organisms to separate from thelipid-containing organism, using at least one hollow fiber contactor.

In some embodiments, the homogenates and extracts may be stabilized bythe addition of one or more stabilizing agents, such as a lipidstabilizing agent, or by packaging in a package designed to preventoxidation. In some embodiments, antioxidants such as vitamin E are addedto the extract to reduce rate of lipid oxidation. In some embodiments,the extracts are packaged in a container under an inert atmosphere. Insome embodiments, the extract is packaged to reduce rate of lipidoxidation in air-free containers such as aluminum coated bags (less than10 kg per bag for efficient removal of oxygen), or containers filledwith nitrogen to remove oxygen. In other embodiments, the extracts arepackaged in vacuum packed containers with a pump delivery system.

In some embodiments, extracts from stem cells, such as embryonic stemcells, are prepared in a like manner. In these embodiments, the stemcells are first disrupted and then centrifuged as above to removeinsoluble cellular debris. The stem cells generally comprise much lesslipid material, so the initial centrifugation yields two main fractions,a pellet and cellular fractions which primarily contains cytoplasmiccomponents. In some embodiments, cells, either a plate of cells or cellscollected from flasks or fermentors, are washed in ice cold PBS. When aplate of cells is utilized, the cells are scraped and transferred to anice cold centrifuge tube, such as a 1.5 ml microfuge tube. In someembodiments, the cells are then pelleted and the supernatant is removed.The cells are then disrupted. In some embodiments, a hypotonic solutionis added to the cells in a volume of from about 1.5:1 to 3.0:1 ascompared to the cell pellet. A suitable hypotonic solution comprises 10mM HEPES pH 7.9, 1.5 mM MgCl2, 10 mM KCl 3.33, 0.5 mM DTT, and 0.2 mMPMSF. In some embodiments, a 10% solution of Triton X is then added(about 1/20 volume) to the pellet and the pellet resuspended byvortexing. In some embodiments, the cells are then homogenized, forexample with a Dounce homogenizer or sonicated to further disrupt thecells. In some embodiment, the cellular debris is then pelleted bycentrifugation, for example 6,000 RPM at 4° C. for 30 seconds. Thesupernatant is then collected as the cellular extract.

In some embodiments, the cellular extracts described above, and mostpreferably the middle fractions, are further fractionated. A variety ofmethod may be used, including, but not limited to, FICOL gradients,gradient centrifugation, protein precipitation, freeze drying, columnchromatography, such as size exclusion chromatography and affinitychromatography, gel separation, high pressure liquid chromatography,ChIP, and immunoprecipitation. It will be recognized that these fractionsteps yield corresponding fractions such as freeze dried fractions,affinity chromatography fractions, precipitated fractions, etc.

In some embodiments, the fractions are then combined with orresolubilized with components suitable for preparing compositions fortopical administration as described in more detail below.

F. Epigenetic Inhibitors

In some embodiments, the compositions of the present invention furthercomprise epigenetic inhibitors. In preferred embodiments, one or moreepigenetic inhibitors are combined with one or more of the cellularextracts described in Sections A-E. The present invention is not limitedto the use of any particular epigenetic inhibitors. Indeed, the use ofvariety of epigenetic inhibitors is contemplated, including, but notlimited to synthetic epigenetic inhibitors and epigenetic inhibitorsisolated or derived from natural sources. Examples of epigeneticinhibitors include, but are not limited to histone deacetylaseinhibitors, DNA methyltransferase inhibitors and some vitamins.

In some embodiments, the epigenetic inhibitors comprises a naturalextract containing butyrate or butyric acid made from natural foods suchas butter from animal fats or milk (e.g. cow's milk or cheese), plantoils (e.g. Heracleum giganteum (cow parsnip) and Pastinaca sativa(parsnip)), or Kombucha tea (includes Butyric Acid as a result offermentation containing butyrate). Extract preparation may includefermentation by obligate anaerobic bacteria (e.g. Clostridium butyricum,Clostridium kluyveri, Clostridium pasteurianum, Fusobacterium nucleatum,Butyrivibrio fibrisolvens, Eubacterium limosum). Animal fat or plant oilproduct extracts may be prepared by chemical or physical processesinducing the liberation of butyric acid from the glyceride byhydrolysis. The extract could also be prepared by the fermentation ofsugar or starch in the natural foods by the addition of Bacillussubtilis, with calcium carbonate added to neutralize the acids formed.

In other embodiments, the epigenetic inhibitors comprise a naturalextract of red grapes containing the phytoalexin resveratrol, includingan extract from juice or fermented juice (wine) of red grapes. Extractscould be prepared by mechanical disruption of grapes, separation of theskin from the flesh and seeds, and either extracting phytoalexin bychemical or mechanical methods, or be prepared from fresh or fermentedgrape juice by chemical or physical methods including boiling,fractionation, affinity chromatography, freeze-drying or gel separation.

In other embodiments, the epigenetic inhibitors comprise a naturalextract containing Cyanocobalamin (vitamin B₁₂) made from organismscontaining enzymes required for the synthesis of B₁₂ such as bacteriaand archaea, or natural products which harbor such B₁₂ producingbacteria including meat (especially liver and shellfish), eggs, and milkproducts. Extracts can be prepared by chemical or physical methods suchas homogenization followed by fractionation, affinity chromatography,freeze-drying or gel separation.

In other embodiments, the epigenetic inhibitors comprise a naturalextract containing one or several variants of vitamin B, made fromeither potatoes, bananas, lentils, chili peppers, tempeh, liver, turkey,tuna, nutritional yeast (or brewer's yeast), beer or marmite. Extractscan be prepared by chemical or physical methods such as homogenizationfollowed by e.g. fractionation, affinity chromatography, freeze-dryingor gel separation.

In other embodiments, the epigenetic inhibitors comprise a naturalextract containing retinoids or retinoid precursors, made from eitheranimal sources (e.g. milk and eggs) which contain retinyl esters, orfrom plants (e.g. carrots, spinach) which contain pro-vitamin Acarotenoids. The extract may be modified by hydrolysis (animal sources)of retinyl esters to result in retinol, while plant extracts containingpro-vitamin A carotenoids can be cleaved to produce retinal(retinaldehyde), which can be further be reversibly reduced to produceretinol or it can be irreversibly oxidized to produce retinoic acid. Thebest described active retinoid metabolites are 11-cis-retinal and theall-trans and 9-cis-isomers of retinoic acid, which may be added to thisextract.

Examples of other DNA methyltransferase inhibitors include, but are notlimited to, 5-Azacytidine, 5-Aza-20-deoxycytidine,Arabinosyl-5-azacytidine, 5-6-Dihydro-5-azacytidine,5-Fluoro-20-deoxycytidine, EGX30P, Epigallocatechin-3-gallate, Green teapolyphenol, Hydralazine, MG98, Procainamide, Procaine, and Zebularine.Examples of other histone deacetylase inhibitors include, but are notlimited to Apicidin, Butyrates, Phenylbutyrate, m-Carboxycinnamic acidbishydroxamide (CBHA), Cyclic hydroxamic-acid-containing peptide 1(CHAP1), TSA-Trapoxin Hybrid, Depudecin Epoxide, Depsipeptide FR901228,Benzamidine, LAQ824, Oxamflatin, MGCD0103, PXD101, Pyroxamide, SubericBishydroxamic Acid (SBHA), Suberoylanilide Hydroxamic Acid (SAHA),Trichostatin A (TSA), Trapoxin A, and Valproic acid.

G. Topical Delivery Methods

In some embodiments, the extracts described above (or components of theextracts) are formulated for topical delivery. General formulations fortopical delivery are described in Remington's Pharmaceutical Sciences,18th Edition, Mack Publishing, p. 1288-1300 [1990]. Accordingly, in someembodiments, the extracts are formulated as a water based gel or paste,ointment, cream (anhydrous or hydrous), lotion (anhydrous or hydrous),emulsion, spray, solution, aerosol, stick (solid cream), liquid bandaid, powder, inhalation spray, nasal spray, basal drops, cheek drops,sublingual drops, eye drops or sprays, ear drops or sprays, andtransdermal patches.

H. Other Delivery Methods

In some embodiments, the extracts described above (or components of theextracts) are formulated for delivery by a variety of methods. In someembodiments, the extracts described above are formulated for delivery toskin, gastrointestinal tractus, fat deposits, cartilage, bone,connective tissue, muscle or internal organs. In some embodiments, theextracts or components thereof are formulated for oral administrationwith or without suitable carriers such as starch, sucrose or lactose intablets, pills, dragees, capsules, solutions, liquids, slurries,suspensions and emulsions. In some embodiments, the oral deliveryvehicle comprises an enteric coating. In other embodiments, the extractsor components thereof are formulated for rectal administration as acapsule, cream, suppository or liquid. In some embodiments, the extractsof components thereof are injected by syringe to the peritoneal cavityor into internal organs or tissues. In some embodiments, the extracts orcomponents thereof are formulated for delivery an osmotic pump.

In still other embodiments, the extracts or components thereof aredelivered by microinjection, preferably via particle bombardment (i.e.,with a gene gun). Particle mediated gene transfer methods are known inthe art, are commercially available, and include, but are not limitedto, the gas driven gene delivery instrument descried in McCabe, U.S.Pat. No. 5,584,807, the entire contents of which are herein incorporatedby reference. This method involves coating the nucleic acid sequence ofinterest onto heavy metal particles, and accelerating the coatedparticles under the pressure of compressed gas for delivery to thetarget tissue. Other particle bombardment methods are also available.Generally, these methods involve depositing the extract or componentsthereof upon the surface of small, dense particles of a material such asgold, platinum, or tungsten. The coated particles are themselves thencoated onto either a rigid surface, such as a metal plate, or onto acarrier sheet made of a fragile material such as mylar. The coated sheetis then accelerated toward the target biological tissue. The use of theflat sheet generates a uniform spread of accelerated particles thatmaximizes the number of cells receiving particles under uniformconditions, resulting in the introduction of the nucleic acid sampleinto the target tissue. This invention contemplates the described use ofgene-gun to deliver extracts or components of extracts as defined above.

In still other embodiments, the embryonic stem cell, adult stem cell oregg extracts or components are microencapsulated (e.g., with collagen orglycosaminoglycans), formed into nanoparticles (e.g., lecithinencapsulated in an oil core), liposomes, microemulsions, ornanoemulsions, oil bodies, retinol molecular fluid films, unilamellarvesicles, multilamellar vesicles, preloaded spherical beads or sponges,elastic vesicles, etc.

I. Composition Profiles

In some embodiments the composition for topical and/or internalapplication is a combination of extracts with lipids and/or water and/orcarbohydrates and/or nucleic acids and/or proteins and/or signalingsubstances. In some embodiments the extract herein is composed of wholecells or a combination of lipids and/or carbohydrates and/or nucleicacids and/or proteins and/or signaling substances of the cells fromwhich the extract is made, or from synthetic and/or natural versions oflipids and/or carbohydrates and/or nucleic acids and/or proteins and/orsignaling substances. Signaling profiles include combinations of activesubstances released from cells which are contained in extracts of cells,and include synthetic and/or natural versions of these signalingsubstances added to extracts. Signaling substances contemplated includebut are not limited to growth factors, endorphins, hormones, amino acidtransmitters, immunoregulatory cytokines and other immunity-associatedfactors.

Transforming growth factor-β1 orchestrates the biology of irradiatedtissue as a tissue level sensor of oxidative stress, and is integral tothe cellular DNA damage response. Transforming growth factor-β5(TGF-β5), a member of this signaling factor family found in amphibians,is expressed in regenerating blastemas formed under limb regeneration(King et al., 2003), and all mammalian isoforms of TGF-β are releasedlocally from various cells at sites of injury and are important in thecontrol of fibrosis and scarring during mammalian tissue repair.Manipulation of specific TGF-β isoforms is capable of producingscar-free healing of wounds in mice (Ferguson and O'Kane, 2004). TGF-β1is a potent immunoregulatory cytokine involved in suppression ofinflammation and regulatory T cell activity, resulting in immunetolerance (Chen and Wahl, 2003). Studies on wound healing andimmunosuppression in mammals indicates that differential activity ofTGF-β in regenerating amphibian limb stumps may be involved suppressionof fibrosis and establishing conditions permissive for blastemaformation.

Transforming growth factor-alpha (TGF-α) and brain-derived neurotrophicfactor (BDNF) secreted in vitro from human pluripotent stem cellsderived from embryonic germ cells, termed embryoid body-derived (EBD)cells, have the capacity to restore neurologic function in animals byprotecting host neurons from death and facilitate reafferentation ofmotor neuron cell bodies (Kerr D A, et al., Human embryonic germ cellderivatives facilitate motor recovery of rats with diffuse motor neuroninjury. J Neurosci. 2003 Jun. 15; 23(12):5131-40).

Fibroblast growth factors (FGFs) such as FGF-10 have been demonstratedto be of importance in regrowth of limbs in frogs (Christen and Slack,1997; Yokoyama et al., 2000).

The Pro-opiomelanocortin (POMC) precursor for a-melanocyte stimulatinghormone (α-MSH), endorphins, and several other peptide hormones, isexpressed in regeneration blastemas (King et al., 2003), in skin as wellas brain, pituitary, and other organs. POMC is a central importance inmodulating immune activity within skin, primarily due to the activity ofα-MSH (Luger et al., 1999). Paracrine release of α-MSH peptides exerts apotent immunomodulatory effect on immune cells. α-MSH inhibits all formsof inflammation against which it has been tested (Lipton et al., 1997)and localized production of α-MSH helps maintain optimal immune responseat specific sites in the skin (Paus et al., 2003). Expression of α-MSHcells of a blastoma would be expected to confer an anti-inflammatoryeffect potentially important for inhibiting fibrosis and regenerationnecessary for limb or tissue regrowth.

Thymosin-β4 is a thymic maturation factor that has also been shown topromote angiogenesis, keratinocyte migration and wound healing (Malindaet al., 1999). Thymosin-β4 exerts potent anti-inflammatory activity andis secreted by macrophages and T lymphocytes of skin, gut and otherorgans in addition to the thymus (Young et al., 1999; Girardi et al.,2003). Thymosin-β4 is up-regulated in frog pseudoblastemas (King et al.,2003) and regenerating blastemas and activities of thymosin-β4 intissues of amputated limbs may include immunomodulation of theinflammatory response in addition to stimulation of epithelial migrationand other aspects of regeneration.

J. Additional Components

In some embodiments, the extracts or components thereof described aboveare combined with additional components. In some embodiments, theseadditional components enhance uptake, bioavailability or penetration ofthe extract components. In preferred embodiments, extract components maycontain natural or a mixture of synthetic components. The components maybe partially or totally synthetic. In some embodiments, the cell orextract or synthetic components made from substances identified in theextracts are mixed with a composition comprising water, sebaceous andepidermal lipids and cell extracts, proteins, and components thereof,preferably comprises about a 10% lipid fraction by weight, about a 10%protein fraction by weight, and about an 80% volatile fraction byweight.

Vernix caseosa (vernix) is a naturally occurring skin protectant. Vernixis a lipid rich substance composed of sebum, epidermal lipids, anddesquamated epithelial cells that progressively covers the skin of thedeveloping fetus, completely surrounded by amniotic fluid, during thelast trimester of pregnancy. In some embodiment, the invention relatesto compositions where the lipid fraction preferably comprises componentsin vernix, i.e., lecithin and other phospholipids, squalene, waxes, waxesters, sterol esters, diol esters, triglycerides, free sterols and fourclasses of fatty acids ranging in chain length from C₁₂ to C₂₆ (straightchain saturated, straight chain unsaturated, branched chain saturated,and branched chain unsaturated). In preferred embodiments, the vernixlipid components are as follow, with the relative percentages indicated,squalene (9%), aliphatic waxes (12%), sterol esters (33%), diesters(7%), triglycerides (26%), free sterols (9%), other lipids (4%). Inadditional embodiments, the lipid composition is composed of lipids fromegg and/or fish roe with wound healing properties 30% of which arebarrier lipids (proteolipid matrix); cholesterol (1.1%, 52.8% ofbarrier), free fatty acids (0.6%, 27.7% of barrier), phospholipids(0.4%), ceramides (0.7%, 20.1% barrier). In another preferredembodiment, the protein fraction contains the protein components ofvernix, i.e., keratin, filaggrin, regulator proteins (e.g., EGF), andglutamine.

The fatty acids within the aliphatic waxes may be branched and thebranched fatty acids may be methylated. The protein fraction consists ofepidermally derived proteins, primarily keratin and filaggrin. Theprotein fraction also contains trace amounts in the range of aboutmicromolar to millimolar concentrations of regulatory proteins such asepidermal growth factor (EGF), and trace amounts of about nanomolar tomicromolar concentrations of surfactant protein such as Surfactant A andSurfactant B. The volatile fraction is primarily water. The rate ofevaporation of volatile components is relatively slow, presumably due toincreased energy requirements for the dissociation of hydrogen bonds andfor diffusion from the cellular component through the lipid component tochange water from the liquid to the gaseous state. In additionalpreferred embodiments, the composition contains mRNA contained in cellextracts, preferably stem cell extracts.

In some embodiments, the embryonic stem cell, adult stem cell or eggextracts or components are combined with phospholipids or otherlipophilic substances, palmitylmyristrates, dimethylsulfoxide (DMSO),chitosan, long chain organic polymers such as polysaccharides,non-aqueous solvents, beta-glucan, pH adjusting components, skinmetabolism inhibition agents, propylene glycol, butylenes glycol,polyethylene glycol, olive oil or other naturally occurring oils,dimethyl isosorbide, dimethylformamide, methyl salicylate, long chainoleic acid, mucopolysaccharides, and other agents.

In some embodiments, the additional agents include, but are not limitedto, ubiquitin, antimicrobial agents (alpha-defensins, LL37,beta-defensins, etc.), surfactant proteins from the collectin family(collecting associated protein A and D), nicotinamide and psoriacin.

In some embodiments, the additional agents include, but are not limitedto, vitamins, antioxidants, minerals, extracts, and chemical compoundssuch as alpha-tocopherol (vitamin E), melanin, vitamin C, provitamin A,retinyl proprionate, retinoic acid, Vitamin D3, Nicotinamide (vitaminB), Niacinaminde (Vitamin B3, exfoliates surface skin), d-panthenol(aids in skin repair of damage), vitamin A, hyaluronic acid, ceramides,Seaweed (algae) Mineral oil (paraffinum liquidium) Petrolatum GlycerinIsohexadecane Cirtus aurantifolia (lime) extract Microcrystalline wax(cera microcristallina) Lanolin alcohol Seamum indicium (sesame) seedoil, Eucalyptus globules (eucalyptus) leaf oil, Magnesium sulfate,Sesamum indicum (sesame) seeds, Medicago satvia (alfalfa) seeds,Helianthus annuus (sunflower) seeds, Prunus dulcis (powdered almonds),Sodium, Potassium, Copper, Calcium, Magnesium, zinc gluconate, Paraffin,Vitamin E succinate, Niacin, Beta-carotene, Decyl oleate, Aluminumdistearate, Octyuldodecanol, Citric acid, Cyanocobalamin, Magnesiumstearate, Panthenol, Limonene, Geraniol, Linalool, Hydroxycitronellal,Citronellol, Benzyl salicylate, Citral, Methylchloroisothiazoline,Methylisothiazolinone, Alcohol denat., Fragrance (parfum), Butyleneglycol, Byrospermum parkii (shea butter), Fish (pisces) cartilageextract, Polyethylene, Hydrogenated polyisobutene, Cyclopentasiloxane,Cetyl esters, Cetearyl alcohol, Malachite, Isostearyl neopentanoate,Polybutene, Sucrose, Silica, Tocotrienol, Cucumis satvius (cucumber)fruit extract, Centella asiatica (hydrocotyl) extract, Seamum indicium(sesame) seeds, Eucalyptus globules (eucalyptus) leaf oil, Medicagosatvia (alfalfa) seeds, Helianthus annuus (sunflower) seeds, Prunusdulcis (powdered almonds), Potassium, Copper, Calcium, Magnesium,Caffeine, Sodiumhyaluronate, Linoleic acidCholesteryl/behenyl/octyldodecyl lauroyl glutamate, Methyl glucosesesquisterate, Cholesterol, Dimethicone, Ocimum basilicum (basil),Mentha arvensis (wild mint), Acrylates/C10-30 alkyl acrylatecrosspolymer, Glyceryl distearate, Cetearyl glucoside, Steareth-10,Carbomer, Aminomethyl propanol, Limonene, Linalool, Benzyl salicylate,Disodium EDTA, BHT, Sodium dehydroacetate, Phenoxyethanol,Methylparaben, Titanium dioxide (CI 77891), C12-20 acid PEG-8 Ester,Hydrogenated vegetable oil, Petrolatum, Butylene Glycol, Glycerin,Acetylated Lanolin, Glycoproteins, Panax, Ginseng Root extract,Equisetum Arvense (Horsetail) Extract, Sodium carbomer, Beeswax (ceraalba), Cetyl phosphate, Polyperfluoromethylisoporpyl ether, Benzylalcohol, Linalool, Hydroxycitronellal, Alpha-isomethyl ionone, Amylcinnamal, Hexyl cinnamal, Verenia furfuracea (treemoss) extract,Geraniol, Benzyl benzoate, Bytulphenol methylpropional, Eugenol, Benzylsalicylate, Chlorphenesin, Phenoxyethanol, and Methylparaben.

In some embodiments, the compositions of the present invention areuseful for facilitating the delivery of active compounds via the skin.In some preferred embodiments, one or more active agents, such as aprotein, small organic compound, or one of the agents identified aboveare combined with the cytoplasmic fraction of, for example, a fertilizedor unfertilized amphibian or fish eggs. Cytoplasmic fractions and methodfor making such fractions are disclosed elsewhere in the application indetail. Accordingly, in some embodiments, the present invention providescompositions comprising a cytoplasmic fraction of amphibian or fish eggsand one or more active agents. In some embodiments, the presentinvention provides methods of facilitating the penetration of one ormore active agents into the skin, comprising providing a compositioncomprising a cytoplasmic extract from amphibian and/or fish eggs and oneor more active agents and contacting the skin of a subject with thecomposition. As described above, the composition can be preferably be anemulsion, salve, cream, gel, spray, aerosol, liquid, etc.

Exemplary proteins that can be active agents include, but are notlimited to, Alzheimer's amyloid peptide (Aβ), SOD1, presenillin 1 and 2,renin, α-synuclein, amyloid A, amyloid P, activin, anti-HER-2, bombesin,enkephalinase, protease inhibitors, therapeutic enzymes, α1-antitrypsin,mammalian trypsin inhibitor, mammalian pancreatic trypsin inhibitor,calcitonin, cardiac hypertrophy factor, cardiotrophins (such ascardiotrophin-1), CD proteins (such as CD-3, CD-4, CD-8 and CD-19),CFTR, CTNF, DNase, human chorionic gonadotropin, mousegonadotropin-associated peptide, cytokines, transthyretin, amylin,lipoproteins, lymphokines, lysozyme, a growth hormone (including humangrowth hormone), bovine growth hormone, growth hormone releasing factor,parathyroid hormone, thyroid stimulating hormone, growth factors,brain-derived neurotrophic growth factor, epidermal growth factor (EGF),fibroblast growth factor (such as α FGF and β FGF), insulin-like growthfactor-I and -II, des(1-3)-IGF-I (brain IGF-I), insulin-like growthfactor binding proteins, nerve growth factor (such as NGF-β),platelet-derived growth factor (PDGF), vascular endothelial growthfactor (VEGF), receptors for growth hormones or growth factors,transforming growth factor (TGF) (such as TGF-α, TGF-β1, TGF-β2, TGF-β3,TGF-β4 or TGF-β5), neurotrophic factors (such as neurotrophin-3, -4, -5,or -6), gelsolin, glucagon, kallikreins, mullerian-inhibiting substance,neurotrophic factors, p53, protein A or D, prorelaxin, relaxin A-chain,relaxin B-chain, rheumatoid factors, rhodopsin, a serum albumin (such ashuman serum albumin), inhibin, insulin, insulin chains, insulin A-chain,insulin β-chain, insulin receptor, proinsulin, luteinizing hormone,integrin, interleukins (ILs) (such as IL-1 to IL-10, IL12, IL-13),erythropoietin, thrombopoietin, fibrillin, follicle stimulating hormone,clotting factors (such as factor VIIIC, factor IX, tissue factor, andvon Willebrands factor, anti-clotting factors (such as Protein C, atrialnaturietic factor, lung surfactant), a plasminogen activator (such ashuman tissue plasminogen activator or urokinase), thrombin, tumornecrosis factor-αor β, α-ketoacid dehydrogenase, addressins, bonemorphogenetic proteins (BMPs), collagen, colony stimulating factors(CSFs) (such as M-CSF, GM-CSF and G-CSF), decay accelerating factor,homing receptors, interferons (such as interferon-α, -β and -γ),keratin, osteoinductive factors, PRNP, regulatory proteins, superoxidedismutase, surface membrane proteins, transport proteins, T-cellreceptors, viral antigens such as a portion of the AIDS envelope,immunoglobulin light chain, antibodies, antibody fragments (such assingle-chain Fv fragment (scFv), single-chain antibody (scAb), F_(AB)antibody fragment, diabody, triabody, fluorobody), antigens such asgp120(IIIb) immunotoxins, atrial natriuretic peptide, seminal vesicleexocrine protein, β2-microglobulin, PrP, precalcitonin, ataxin 1, ataxin2, ataxin 3, ataxin 6, ataxin 7, huntingtin, androgen receptor,CREB-binding protein, gp120, p300, CREB, AP1, ras, NFAT, jun, fos,dentaorubral pallidoluysian atrophy-associated protein, a microbialprotein (e.g., maltose binding protein, ABC transporter, glutathione Stransferase, thioredoxin, β-lactamase), green fluorescent protein, redfluorescent protein, or derivatives or active fragments or geneticvariants of any of the peptides listed above.

Examples of small organic compounds include, but are not limited to,non-steroidal anti-inflammatory drugs (NSAIDS) (the NAIDS can, forexample, be selected from the following categories: (e.g., propionicacid derivatives, acetic acid derivatives, fenamic acid derivatives,biphenylcarboxylic acid derivatives and oxicams)); steroidalanti-inflammatory drugs including hydrocortisone and the like;antihistaminic drugs (e.g., chlorpheniranune, triprolidine); antitussivedrugs (e.g., dextromethorphan, codeine, carmiphen and carbetapentane);antipruritic drugs (e.g., methidilizine and trimeprizine);anticholinergic drugs (e.g., scopolamine, atropine, homatropine,levodopa); anti-emetic and antinauseant drugs (e.g., cyclizine,meclizine, chlorpromazine, buclizine); anorexic drugs (e.g.,benzphetamine, phentermine, chlorphentermine, fenfluramine); centralstimulant drugs (e.g., amphetamine, methamphetamine, dextroamphetamineand methylphenidate); minoxidil; antiarrhythmic drugs (e.g., propanolol,procainamide, disopyraminde, quinidine, encainide); P-adrenergic blockerdrugs (e.g., metoprolol, acebutolol, betaxolol, labetalol and timolol);cardiotonic drugs (e.g., milrinone, amrinone and dobutamine);antihypertensive drugs (e.g., enalapril, clonidine, hydralazine,minoxidil, guanadrel, guanethidine); diuretic drugs (e.g., amiloride andhydrochlorothiazide); vasodilator drugs (e.g., diltazem, amiodarone,isosuprine, nylidrin, tolazoline and verapamil); vasoconstrictor drugs(e.g., dihydroergotamine, ergotamine and methylsergide); antiulcer drugs(e.g., ranitidine and cimetidine); anesthetic drugs (e.g., lidocaine,bupivacaine, chlorprocaine, dibucaine); antidepressant drugs (e.g.,imipramine, desipramine, amitryptiline, nortryptiline); PDE5 inhibitorssuch as Viagra® or Cialis®; tranquilizer and sedative drugs (e.g.,chlordiazepoxide, benacytyzine, benzquinamide, flurazapam, hydroxyzine,loxapine and promazine); antipsychotic drugs (e.g., chlorprothixene,fluphenazine, haloperidol, molindone, thioridazine and trifluoperazine);antimicrobial drugs (antibacterial, antifungal, antiprotozoal andantiviral drugs).

Antimicrobial drugs which are preferred for incorporation into thepresent composition include, for example, pharmaceutically acceptablesalts of β-lactam drugs, quinolone drugs, ciprofloxacin, norfloxacin,tetracycline, erythromycin, amikacin, triclosan, doxycycline,capreomycin, chlorhexidine, chlortetracycline, oxytetracycline,clindamycin, ethambutol, hexamidine isothionate, metronidazole;pentamidine, gentamycin, kanamycin, lineomycin, methacycline,methenamine, minocycline, neomycin, netilmycin, paromomycin,streptomycin, tobramycin, miconazole, and amanfadine.

Other drug moieties of use in practicing the present invention includeantineoplastic drugs (e.g., antiandrogens (e.g., leuprolide orflutamide), cytocidal agents (e.g., adriamycin, doxorubicin, taxol,cyclophosphamide, busulfan, cisplatin, a-2-interferon) anti-estrogens(e.g., tamoxifen), antimetabolites (e.g., fluorouracil, methotrexate,mercaptopurine, thioguanine).

The compositions can also comprise hormones (e.g., medroxyprogesterone,estradiol, leuprolide, megestrol, octreotide or somatostatin); musclerelaxant drugs (e.g., cinnamedrine, cyclobenzaprine, flavoxate,orphenadrine, papaverine, mebeverine, idaverine, ritodrine,dephenoxylate, dantrolene and azumolen); antispasmodic drugs;bone-active drugs (e.g., diphosphonate and phosphonoalkylphosphinatedrug compounds); endocrine modulating drugs (e.g., contraceptives (e.g.,ethinodiol, ethinyl estradiol, norethindrone, mestranol, desogestrel,medroxyprogesterone), modulators of diabetes (e.g., glyburide orchlorpropamide), anabolics, such as testolactone or stanozolol,androgens (e.g., methyltestosterone, testosterone or fluoxymesterone),antidiuretics (e.g., desmopressin) and calcitonins).

Also of use in the present invention are estrogens (e.g.,diethylstilbesterol), glucocorticoids (e.g., triamcinolone,betamethasone, etc.) and progenstogens, such as norethindrone,ethynodiol, norethindrone, levonorgestrel; thyroid agents (e.g.,liothyronine or levothyroxine) or anti-thyroid agents (e.g.,methimazole); antihyperprolactinemic drugs (e.g., cabergoline); hormonesuppressors (e.g., danazol or goserelin), oxytocics (e.g.,methylergonovine or oxytocin) and prostaglandins, such as mioprostol,alprostadil or dinoprostone, can also be employed.

Other useful active compounds include immunomodulating drugs (e.g.,antihistamines, mast cell stabilizers, such as lodoxamide and/orcromolyn, steroids (e.g., triamcinolone, beclomethazone, cortisone,dexamethasone, prednisolone, methylprednisolone, beclomethasone, orclobetasol), histamine H₂ antagonists (e.g., famotidine, cimetidine,ranitidine), immunosuppressants (e.g., azathioprine, cyclosporin), etc.Groups with anti-inflammatory activity, such as sulindac, etodolac,ketoprofen and ketorolac, are also of use. Other drugs of use inconjunction with the present invention will be apparent to those ofskill in the art.

In some embodiments, components of the extract may act as chemotaxants.Mesenchymal stem cells and fibrocytes circulates in the blood stream andin case of skin wound they penetrate the wound area where they candifferentiate to skin cells like fibroblasts, keratinocytes, pericytes,adipose and endothelial cells. Chemotaxants in the extract may act asligands for the CCR7 involved in attractin immune cells and dendriticcells and may include SLC/6Ckine/Exodus2/TCA4 andCKbeta-11/MIP-3beta/ELC

K. Topical Application

It is contemplated that the compositions for topical applicationdescribed above find use for both cosmetic and therapeutic purposes.Therapeutic uses are described in more detail in Section J. In someembodiments, it is contemplated that the compositions described aboveare applied directly to the skin or other epithelial or epidermalsurfaces of the body. The compositions may be applied one, two, three ormore times each day as is appropriate for the indication. The amountapplied is not generally important, but generally a compositioncomprising from about 0.001 μg to 10 grams of the extract (or componentsthereof) may be applied to a given surface of the body. As describedabove, the composition may comprise other components such as adjuvants,carriers, other active ingredients, etc.

In some embodiments, the invention relates to compositions that includepreservatives and antioxidants (including vitamins) to prevent productdeterioration preferably trisodium and tetrasodium edetate (EDTA) andtocopherol (vitamin E). In further embodiments the composition containsantimicrobials to fight bacteria preferably butyl, propyl, ethyl, andmethyl parabens, DMDM hydantoin, methylisothiazolinone phenoxyethanol(also rose ether fragrance component), quaternium-15. In furtherembodiments, the composition contains thickeners and waxes used in stickproducts such as lipsticks and blushers preferably candelilla, carnauba,and microcrystalline waxes carbomer and polyethylene—thickeners. Infurther embodiments, the composition contains solvents to dilutepreferably butylene glycol and propylene glycol, cyclomethicone(volatile silicone), ethanol (alcohol) and glycerin. In furtherembodiments, the composition contains emulsifiers to break up and refinepreferably glyceryl monostearate (also pearlescent agent), lauramide DEA(also foam booster) and polysorbates. In some embodiments, thecompositions contain color additives—synthetic organic colors derivedfrom coal and petroleum sources preferably D&C Red No. 7 Calcium Lake(and other dyes that do not dissolve in water), iron oxides, mica(iridescent), and aminophenols. In further embodiments, the compositionscontain pH adjusters to stabilize or adjust acids and bases preferablyammonium hydroxide—in skin peels and hair waving and straightening,citric acid—adjusts pH, and triethanolamine—pH adjuster used mostly intransparent soap. In further embodiments, the compositions containsagents preferably magnesium aluminum silicate—absorbent, anti-cakingagent, silica (silicon dioxide)—absorbent, anti-caking, abrasive, sodiumlauryl sulfate—detergent, stearic acid—cleansing, emulsifier, talc(powdered magnesium silicate)—absorbent, anti-caking, and zincstearate—used in powder to improve texture, lubricates.

The composition includes the recited components and combinations thereofin a total amount of about 0.5 to 50 grams per liter, preferably about 3to 10 grams per liter, although higher or lower concentrations arepermissible. Such compositions being in the form of an emulsion, cream,salve or the like, the active materials being admixed with water,alkylene glycols, various oils natural and synthetic, petrolatum,preservatives, coloring agents, perfumes, and like ingredientsconventional in the cosmetic arts.

The composition can be applied to the face, eyelids or other body partsin an amount varying with the individual. About 0.01 to 1,advantageously about 0.02 to 0.75 and preferably about 0.3 to 0.5, gramsper cm² has been found useful but more or less can be used. Theapplication can be once weekly or more often, even several times a day.

In accordance with the compositions and method of the present invention,the egg, embryo or stem cell extracts of the present invention may beadministered in the form of a pharmaceutical composition additionallycomprising a pharmaceutically acceptable carrier. One skilled in the artwill appreciate that suitable methods of administering the extractcompositions to an animal, such as a mammal, are available and, althoughmore than one method can be used to administer a particular composition,a particular method and dosage can provide a more immediate and moreeffective reaction than others. Pharmaceutically acceptable carriers arealso well known to those skilled in the art. The choice of carrier willbe determined, in part, both by the particular composition and by theparticular method used to administer the composition. Accordingly, thereis a wide variety of suitable formulations of the pharmaceuticalcompositions of the present invention.

In some preferred embodiments, the formulations of this invention aredesigned for topical administration. Typical of such formulations areointments, creams, and gels.

Ointments generally are prepared using either (1) an oleaginous base,i.e., one consisting of fixed oils or hydrocarbons, such as whitepetrolatum or mineral oil, or (2) an absorbant base, i.e., oneconsisting of an anhydrous substance or substances which can absorbwater, for example, anhydrous lanolin. Customarily, following formationof the base, whether oleaginous or absorbent, the active ingredient(e.g., salmon egg extract or stem cell extract) is added in an amountaffording the desired concentration.

Creams are oil/water emulsions. They consist of an oil phase (internalphase), comprising typically fixed oils, hydrocarbons, and the like,such as waxes, petrolatum, mineral oil, and the like, and an aqueousphase (continuous phase), comprising water and any water-solublesubstances, such as added salts. The two phases are stabilized by use ofan emulsifying agent, for example, a surface active agent, such assodium lauryl sulfate; hydrophilic colloids, such as acacia colloidalclays, veegum, and the like. Upon formation of the emulsion, the activeingredient (e.g., salmon egg extract or stem cell extract) customarilyis added in an amount to achieve the desired concentration.

Gels comprise a base selected from an oleaginous base, water, or anemulsion-suspension base. To the base is added a gelling agent whichforms a matrix in the base, increasing its viscosity. Examples ofgelling agents are hydroxypropyl cellulose, acrylic acid polymers, andthe like. Customarily, the active ingredient (IGF-II) is added to theformulation at the desired concentration at a point preceding additionof the gelling agent.

The amount of extract incorporated into the formulation of thisinvention is not critical; the concentration should only be in a rangesufficient to permit ready application of the formulation to the woundarea in an amount which will deliver the desired amount of extract.

The customary amount of formulation to be applied will depend uponconcentration of the active ingredient in the formulation. In someembodiments, the amount of protein in the extract is determined. Then, aspecific amount of the extract is included in the pharmaceuticallyacceptable carrier based on the amount of protein. Generally, theformulation will be applied to the wound in an amount affording fromabout 0.1 to about 500 μg of protein per cm² of skin. Preferably, theapplied amount of protein will range from about 1 to about 300 μg/cm²,more preferably, from about 5 to about 200 μg/cm². In other embodiments,a specific volume of extract is added to the pharmaceutically acceptablecarrier. Accordingly, in some embodiments, the compositions of thepresent invention comprise on a volume/volume basis (volume of extractand volume of pharmaceutically acceptable carrier), for example, fromabout 0.001 to 50% extract, about 0.01 to 50% extract, about 0.1 to 50%extract, about 0.001 to 10% extract, about 0.01 to 10% extract, about0.1 to 10% extract, about 0.001 to 5% extract, about 0.01 to 5% extract,about 0.1 to 5% extract, about 0.001 to 4% extract, about 0.01 to 4%extract, about 0.1 to 4% extract, about 0.001 to 2% extract, about 0.01to 2% extract, about 0.1 to 2% extract, about 0.001 to 1% extract, about0.01 to 1% extract, or about 0.1 to 1% extract.

The present invention may be formulated as necessary with additives usedcommonly in the pharmaceutical sciences, such as surfactants, oils andfats, polyhydric alcohols, lower alcohols, thickening agents, UVabsorbents, light scattering agents, preservatives, antioxidants,antibiotics, chelating agents, pH regulators, flavoring agents, pigmentsand water.

Examples of surfactants include polyoxyethylene (hereinafter abbreviatedas POE-branched alkyl ethers such as POE-octyldodecyl alcohol andPOE-2-decyltetradecyl alcohol, POE-alkyl ethers such as POE-oleylalcohol ether and POE-cetyl alcohol ether, sorbitan esters such assorbitan monooleate, sorbitan monoisostearate and sorbitan monolaurate,POE-sorbitan esters such as POE-sorbitan monooleate, POE-sorbitanmonoisostearate and POE-sorbitan monolaurate, fatty acid esters ofglycerol such as glyceryl monooleate, glyceryl monostearate and glycerylmonomyristate, POE-fatty acid esters of glycerol such as POE-glycerylmonooleate, POE-glyceryl monostearate and POE-glyceryl monomyristate,POE-dihydrocholesterol ester, POE-hardened castor oil, POE-hardenedcastor oil fatty acid esters such as POE-hardened castor oilisostearate, POE-alkylaryl ethers such as POE-octylphenol ether,glycerol esters such as glycerol monoisostearate and glycerolmonomyristate, POE-glycerol ethers such as POE-glycerol monoisostearateand POE-glycerol monomyristate, polyglycerol fatty acid esters such asdiglyceryl monostearate, decaglyceryl decastearate, decaglyceryldecaisostearate and diglyceryl diisostearate and other nonionicsurfactants; potassium salts, sodium salts, diethanolamine salts,triethanolamine salts, amino acid salts and other salts of higher fattyacids such as myristic acid, stearic acid, palmitic acid, behenic acid,isostearic acid and oleic acid, the above alkali salts of ethercarboxylic acids, salts of N-acylamino acids, N-acylsalconates, higheralkylsulfonates and other anionic surfactants; alkylamine salts,polyamine, aminoalcohol fatty acids, organic silicone resin, alkylquaternary ammonium salts and other cationic surfactants; and lecithin,betaine derivatives and other amphoteric surfactants.

Examples of oils and fats include vegetable oils and fats such ascastor-oil, olive oil, cacao oil, camellia oil, coconut oil, wood wax,jojoba oil, grape seed oil and avocado oil; animal oils and fats such asmink oil and egg yolk oil; waxes such as beeswax, whale wax, lanolin,carnauba wax and candelilla wax; hydrocarbons such as liquid paraffin,squalene, microcrystalline wax, ceresine wax, paraffin wax and vaseline;natural or synthetic fatty acids such as lauric acid, myristic acid,stearic acid, oleic acid, isostearic acid and behenic acid; natural orhigher alcohols such as cetanol, stearyl alcohol, hexyldecanol,octyldecanol and lauryl alcohol; and esters such as isopropyl myristate,isopropyl palmitate, octyldodecyl myristate, octyldodecyl oleate andcholesterol oleate.

Examples of polyhydric alcohols include ethylene glycol, polyethyleneglycol, propylene glycol, 1,3-butyrene glycol, 1,4-butyrene glycol,dipropylene glycol, glycerol, diglycerol, triglycerol, tetraglycerol andother polyglycerols, glucose, maltose, maltitose, sucrose, fructose,xylitose, sorbitol, maltotriose, threitol and erythritol.

Examples of thickening agents include naturally-occurring high molecularsubstances such as sodium alginate, xanthene gum, aluminum silicate,quince seed extract, gum tragacanth, starch, collagen and sodiumhyaluronate; semi-synthetic high molecular substances such as methylcellulose, hydroxyethyl cellulose, carboxymethyl cellulose, solublestarch and cationized cellulose; and synthetic high molecular substancessuch as carboxyvinyl polymer and polyvinyl alcohol.

Examples of UV absorbents include p-aminobenzoic acid, 2-ethoxyethylp-methoxycinnamate, isopropyl p-methoxycinnamate,butylmethoxybenzoylmethane,glyceryl-mono-2-ethylhexanoyl-di-p-methoxybenzophenone, digalloyltrioleate, 2,2′-dihydroxy-4-methoxybenzophenone,ethyl-4-bishydroxypropylaminobenzoate,2-ethylhexyl-2-cyano-3,3′-diphenyl acrylate, ethylhexylp-methoxycinnamate, 2-ethylhexyl salicylate, glyceryl p-aminobenzoate,homomethyl salicylate, methyl o-aminobenzoate,2-hydroxy-4-methoxybenzophenone, amyl p-dimethylaminobenzoate,2-phenylbenzoimidazole-5-sulfonic acid and2-hydroxy-4-methoxybenzophenone-5-sulfonic acid.

Examples of preservatives include benzoates, salicylates, sorbates,dehydroacetates, p-oxybenzoates, 2,4,4′-trichloro-2′-hydroxydiphenylether, 3,4,4′-trichlorocarbanilide, benzalkonium chloride, hinokitiol,resorcinol and ethanol.

Examples of antioxidants include tocopherol, ascorbic acid,butylhydroxyanisole, dibutylhydroxytoluene, nordihydroguaiaretic acidand propyl gallate.

Examples of chelating agents include sodium edetate and sodium citrate.

Examples of antibiotics include penicillin, neomycin, cephalothin,potassium permanganate, selenium sulfide, erythromycin, bacitracin,tethacyclin, chloramphenicol, vancomycin, nitrofurantoin, acrisorcin,chlorodontoin, and flucytosine.

Some of these additives function to enhance the efficacy of thecomposition by increasing the stability or percutaneous absorbability ofthe essential components of the present invention.

Also, any dosage form is acceptable, whether in solution, emulsion,powder dispersion, or others. Applicability is wide, includingfundamental dosage forms such as lotions, emulsions, creams and gels.

In addition to those stated above, suitable vehicles, carriers andadjuvants include water, vaseline, petrolatum, mineral oil, vegetableoil, animal oil, organic and inorganic waxes, polymers such asxanthanes, gelatin, cellulose, collagen, starch, kaolin, carrageenan,gum arabic, synthetic polymers, alcohols, polyols, and the like. Thecarrier can also include sustained release carrier such as lypizomes,microsponges, microspheres, or microcapsules, aqueous base ointments,water in oil or oil in water emulsions, gels or the like.

The dose administered to an animal, particularly a human, in the contextof the present invention should be sufficient to effect a therapeuticresponse over a reasonable time frame. The dose will be determined bythe strength of the particular compositions employed and the conditionof the person. The size of the dose and the frequency of applicationalso will be determined by the existence, nature, and extent of anyadverse side effects that may accompany the administration of aparticular composition.

L. Therapeutic Uses

In some embodiments, the cell or extract compositions are useful forhydration (i.e., treating intravascular dehydration and edema in awounds), waterproofing (i.e., compensate for hypovolemia in wounds),guarding against infection (i.e., protecting wound against infections),protection against oxidation (i.e., prevention of oxygen-free radicalproduction during inflammatory reactions of ischemic tissue), woundhealing (i.e., increased metabolism to aid in hypoxic conditionsespecially of burned skin or cells in anaerobic metabolism). In somepreferred embodiments, the compositions are odorless (i.e.,characterized by an absence of volatile carbon or nitrogen containingcompounds).

In some embodiments, the invention relates to methods of using aplurality of compositions. In preferred embodiments, a first cream isused to loosen and/or dissolve cars by collagen dissolving agents oracids. A second cream with extracts or components thereof as well asother wound healing substances as described herein. In another preferredembodiment a laser, chemical peel, razor, acid, freezing, exfoliantand/or abrasive is used to remove scars or wrinkles followed byapplication of a cream with extracts or components thereof as well asother wound healing substances as described herein.

In some embodiments, the invention relates to a first compositionpreferably a cream that slows wound healing, reduces inflammation,and/or reduces scab formation. This composition is applied for severaldays. In preferred embodiments, the composition comprises a combinationof one or more of anti-inflammatory agents, antihistamines, an extractcomponent or components capable of dampening neutrophil migration and/orproliferation, an extract component or components the stimulatemacrophages, phospholipases, arachidonic acid. In further embodiments,there is a water, lipid, protein content that provides vernix propertiesin the composition. In further embodiments, the components in thecomposition reduce activity of fibrogen cytokines. Preferably, the firstcomposition is applied for about 1 to 3 days.

In another embodiment, the invention relates to a second compositionpreferably a cream that heals wounds by stimulating needed cells.Preferably this composition is applied to a subject after the firstcomposition is applied. Preferably, the second composition is appliedfor about 3 to 14 days. This second composition contains components ofcell and cell extracts that regulate collagenases, activateplasminogenases for clot dissolution, stimulate epithelializaiton,(i.e., migration, proliferation, dedifferentiation, redifferentiation),activate fibronectin and fibroblast growth factors, stimulateangiogenesis, reduce activity of fibrogenic cytokines and regulate genessuch as TP53.

In another embodiment, the invention relates to a third compositionpreferably a cream. Preferably this composition is applied to a subjectafter the application of the second composition. This third compositionfunctions to control collagen remodeling by collagen synthesis anddestruction preferably by collegenases and metalloproteins andpreferably collagen I and inactivate fibronectin, hyaluronic acid andglycosaminoglycans, and dehydrate swelling. The third composition ispreferably applied for about one to six weeks, following the applicationof the first and second compositions. In some embodiments, a matrix isprovided, such as a chitosan matrix, biodegradable polymer matrix,collagen matrix, or liquid band aid.

In some embodiments, the cell and/or extract composition is dispersed ina biocompatible liquid was applied to a physiologically acceptablesupport structure in a liquid state to form a film. A film is definedherein as a surface and/or interfacial covering, in either a liquid or asolid state, with temperature-dependant properties. Film-formingtechniques include but are not limited to spraying, extruding, blowing,pouring, evaporating, coating and painting. The dispersion is presentedas droplets that coalesce to form a film upon encountering the support.

In an alternate embodiment, a preformed film is applied to a support.The physiologically acceptable support structure is one that canwithstand sterilization, preferably by standard sterilization techniquesknown to one skilled in the art such as exposure to gamma radiation,autoclaving, and so on. The support structure is not limited to aparticular composition or configuration and, depending upon its use, mayor may not be sterilized and may take various forms.

In another embodiment, the film is used to enhance skin cell maturationand may be applied to structures such as filters, membranes, beads,particles, and so on. Similarly, the support structure is not limited toa particular state of matter and may be a solid, a semi-solid, a gel andso on. In one embodiment, the support consists of a nylon monofilamentinterpositional surfacing material such as Interfaces pads (WinfieldLaboratories, Inc., Dallas Tex.), Biobrane II™. (Sterling Drug Inc., NewYork, N.Y.) or circular nylon filters of suitable porosity (MicronSeparations Inc., Westboro, Mass.). Other support materials, however,could also be used to practice the invention.

In another embodiment, the film is used to treat or prevent injury dueto substance exposure or trauma, and may be applied to various materialsfor placement either in direct contact or indirect contact with anexposed skin site. The skin site may be intact (e.g., normal skin) ormay be compromised, defined as skin that is damaged or that lacks atleast some of the stratum corneum (e.g., skin damaged by exposure to theagent in question, another agent, the presence of a pathologicalcondition such as a rash or contact dermatitis, a physical trauma suchas a cut, wound, or abrasion, a underdeveloped skin such as occurs in apreterm infant, conditions in which either all or part of the epidermisis exposed, conditions in which part of the dermis has been removed suchas partial thickness wounds encountered in resurfacing procedures suchas chemical peels, dermabrasions, and laser resurfacing, etc.).

The support structure may be permeable to physical and/or chemicalagents, and may take a variety of forms, depending upon its purpose andthe extent of the area requiring dressing or treatment. The film may beapplied to various synthetics such as thermoplastic films, blown filmsand breathable films, and various natural and synthetic fabriccompositions such as woven, non-woven, spun, and stitched fabrics. Theinvention may be used in a variety of products, examples of whichinclude wound dressings and coverings such as bandages, tapes, gauze,adhesive products applied for a short or long term to the skin, ostomycare products, hospital pads such as incontinent pads, absorbent pads,and examination pads, disposable and cloth diapers, and feminine hygieneproducts such as intralabial devices.

In some embodiments, the invention relates to regeneration of thefunction of skin with a desired cosmetic appearance and the preventionof skin damage. In further embodiments, early scar formation isprevented by application of a scar prevention composition when the woundis formed. In further embodiments, stimulating the rejuvenation andregeneration of stressed and aging skin prevents wrinkle formation. Infurther embodiments, the product is applied intermittently to slow thecontinual damage process that occurs as skin ages.

The skin has two main layers, the epidermis and dermis. Below these is alayer of subcutaneous (‘under the skin’) fat. The outer surface of theskin is the epidermis, which itself contains several layers, the basalcell layer, the spinous layer, the granular cell layer, and the stratumcorneum. The deepest layer of the epidermis is the basal cell layer.Here cells are continually dividing to produce plump new skin cells.These cells move towards the skin surface, pushed upward by the dividingcells below them. Blood vessels in the dermis, which is below the basalcell layer, supply nutrients to support this active growth of new skincells. As the basal cells move upwards and away from their blood supply,their cell content and shape change. Cells above the basal cell layerbecome more irregular in shape and form the spinous layer. Above this,cells move into the granular layer. Being distant from the blood supplyin the dermis, the cells begin to die and accumulate a substance calledkeratin.

The stratum corneum (‘horny layer’) is the top layer of the epidermis—itis the layer of the skin that we see from the outside. Cells here areflat and scale-like (‘squamous’) in shape. These cells are dead, containa lot of keratin and are arranged in overlapping layers that impart atough and waterproof character to the skin's surface. Dead skin cellsare continually shed from the skin's surface. This is balanced by thedividing cells in the basal cell layer, thereby producing a state ofconstant renewal. Also in the basal cell layer are cells that producemelanin. Melanin is a pigment that is absorbed into the dividing skincells to help protect them against damage from sunlight (ultravioletlight). The amount of melanin in your skin is determined by geneticmakeup and one's exposure to sunlight. The more melanin pigment present,the darker the color of your skin.

Below the epidermis is the layer called the dermis. The top layer of thedermis, the one directly below the epidermis, has many ridges calledpapillae. On the fingertips, the skin's surface follows this pattern ofridges to create our individual fingerprints. The dermis contains avariable amount of fat, and also collagen and elastin fibers thatprovide strength and flexibility to the skin. In an older person theelastin fibers fragment and much of the skin's elastic quality is lost.This, along with the loss of subcutaneous fat, results in wrinkles.Blood vessels supply nutrients to the dividing cells in the basal layerand remove any waste products. They also help maintain body temperatureby dilating and carrying more blood when the body needs to lose heatfrom its surface; they narrow and carry less blood when the body needsto limit the amount of heat lost at its surface. The skin also containsa number of nerves and glands.

Overall skin quality and appearance can be affected by a variety ofdisorders, including aging, photoaging, acne, enlarged pores, andscarring. The intrinsic process of chronological aging results fromthinning of the epidermis and dermis and loss of elasticity. Thisprocess affects all layers of the face, including subcutaneous tissue,the musculofascial system, the superficial musculoaponeurotic system,and the facial skeleton. The result is bony resorption, atrophy ofsubcutaneous fat, attenuation of the musculofibrous system, andalterations of skin surface. The dermal-epidermal junction flattens,which results in loss of rete ridges and a thinner appearance to theepidermis. The dermis also becomes thin, with a decrease in elasticfibers, collagen production, vascularity, and ground substance. Thebiochemical alterations in collagen and elastin result in a dermis thatis more lax yet less elastic and resilient. Collectively, these changesresult in fine wrinkling of the skin and sagging of the tissues thatoverlay the facial skeleton.

In some embodiments, the invention relates to composition comprisingextracts that can stimulate skin cells to regenerate spontaneously. Inadditional embodiments, cells with elongated telomeres made in situ fromthe subject's own cells are reintroduced to the subject.

Many modalities can resurface the skin to improve skin quality, reduceage spots, soften fine lines, and treat acne or other scars. Modalitiesinclude traditional dermabrasion, chemical peeling, laser resurfacing,and microdermabrasion. The techniques attempt to remove the outer layersof skin with the idea that stimulating new growth will improveappearance. The initial evaluation of skin condition is typicallyaccomplished using Fitzpatrick's scale of sun-reactive skin types, whichdenotes patients' reactions to ultraviolet radiation and existing degreeof pigmentation. Type I patients always burn and never tan. Type IIpatients tan only with difficulty and usually burn. Type III patientstan but sometimes burn. Type IV patients rarely burn and tan with ease.Type V patients tan very easily and very rarely burn. Type VI patientstan very easily and never burn.

Chemical peeling is the chemical removal of layers of skin to improvedermatologic defects. The mechanism of action of peeling agents isrelatively straightforward. Stronger agents such as phenol (with variousadditives such as croton oil and glycerin) and trichloroacetic acid(TCA) produce a chemical necrosis of the skin to variable depths,depending on a number of controlled and uncontrolled variables. Theweaker agents change the pH sufficiently to cause a superficial shock tothe cells and, depending on many variables, cell injury or death. Whenused with a moisturizer, the acid acts simply to cause cellular andintercellular swelling and plumping, leading to transient increase incell and matrix size and lessening of fine lines and rhytides.Sequential treatments lead to exfoliation and a smoother complexion.Continued irritation can lead to many of the same effects of tretinoinor retinoid treatment (i.e., increased thickness of dermis, increasedblood flow to skin). The phenol peel “The Baker formula” is phenol USP88% 3 cm³ 49%; distilled water 2 cm³ 44%; croton oil 3 drops 2.1%; andSeptisol 8 drops 4.5%.

The microdermabrasion technique abrades the skin with a high-pressureflow of crystals. Microdermabrasion is most effective with superficialskin conditions because it produces a superficial depth of injury.Superficial skin conditions include early photoaging, fine lines, andsuperficial scarring. Microdermabrasion is accomplished by placing theskin under tension so that an effective vacuum is achieved. Typically,stretching the treatment area with the nondominant hand and using thedominant hand to guide the handpiece is the method used to achieve thiseffect. When treating the neck, the neck is placed in extension toassist in skin tension. The handpiece is moved over the treatment areain a single, smooth stroke, which can then be repeated. The pressure ofthe crystal stream is controlled with a foot pedal. Thicker skin, suchas that on the forehead, chin, and nose, can be treated moreaggressively (i.e., adjust the speed of handpiece movement or number ofpasses). Decrease the pressure when treating the thinner skin of thelower eyelids and upper cheek. Vertically orient all strokes whentreating the neck.

Laser skin resurfacing (LSR) can be performed as an isolated procedureor as an adjunct to procedures such as transconjunctival blepharoplasty(TCB), facelift, and endoscopic browlift. The laser allows for precisecontrol of ablation depth, and it permits the surgeon to vary thesedepths as needed. In addition to such precision, LSR causes favorableheating of the dermis, which tightens collagen fibers and stimulatesneocollagen secretion by fibroblasts. Two laser wavelengths arepreferred for facial skin resurfacing: pulsed carbon dioxide anderbium:yttrium-aluminum-garnet (Er:YAG). Each Er:YAG pulse removes only25-30 micrometers of tissue compared to the pulsed carbon dioxide, whichremoves 50-100 micrometers. The Er:YAG produces less collateral dermalenergy because the thermal conduction is approximately 5 micrometers;pulsed carbon dioxide is 30-50 micrometers. The laser output of Er:YAGis directly absorbed by collagen and dermal proteins, whereas the carbondioxide laser vaporizes extracellular water in the dermis. Each Er:YAGpass generates the same amount of ablation, whereas the pulsed carbondioxide generates a decreased vaporization depth with each pass.

The composition of the present invention also finds use in woundhealing. A wound is a break in the skin (the outer layer of skin iscalled the epidermis). Wounds are usually caused by cuts or scrapes.Healing is a response to the injury that sets into motion a sequence ofevents. With the exception of bone, all tissues heal with some scarring.The object of proper care is to minimize the possibility of infectionand scarring.

Pressure ulcers are chronic wounds caused by unrelieved pressure thatresults in tissue damage. The ulcers are staged from I to IV, accordingto the level of tissue damage observed. Pressure ulcers are most commonin hospitalized patients, nursing home patients and those with spinalcord injuries. The standard of care for pressure ulcers includesinterval dressing changes, pressure relief, repositioning, physicalstrengthening, nutritional support and infection management. If thewound becomes severe, surgical interventions include wound debridementand skin-flap, muscle-flap or free-flap reconstruction.

The present invention also finds use for the treatment of various skindisorders. Uneven skin, discoloration, and growths can be caused by avariety of factors including genetics, exposure to sun, and/or use ofmedications. Callus formation (Clavus) is a thickening of the skin dueto intermittent pressure and frictional forces. The shape of the handsand feet are important in clavus formation. Specifically, the bonyprominences of the metacarpophalangeal and metatarsophalangeal jointsoften are shaped in such a way as to induce overlying skin friction. Asclavus formation ensues, friction against the footwear is likely toperpetuate hyperkeratosis. Toe deformity, including contractures andclaw, hammer, and mallet-shaped toes, may contribute to pathogenesis.Bunionettes, i.e., callosities over the lateral fifth metatarsal head,may be associated neuritic symptoms due to compression of the underlyinglateral digital nerves. Furthermore, Morton toe, in which the second toeis longer than the first toe, occurs in 25% of the population; this maybe one of the most important pathogenic factors in a callus of thecommon second metatarsal head, i.e., an intractable plantar keratosis.

Moles (Nevi) are nests of melanocytes that are in contact with eachother. They typically start formation during early childhood. It hasbeen suggested that they form in response to sun exposure. However, agenetic factor is clearly involved in nevi. Some kinships express anautosomal dominant condition in which members have a large number oflarge nevi, sometimes more than 150 nevi scattered over the integument.Nevi have been observed to develop rapidly after blistering events, suchas second-degree thermal burns or sunburns; toxic epidermal necrolysis;and in persons with genetic blistering diseases, such as epidermolysisbullosa. Growth factors, such as basic fibroblast growth factor, havebeen suggested to be released by proliferation keratinocytes and tostimulate melanocyte proliferation. Melanocytic nevi are benignneoplasms or hamartomas composed of mostly melanocytes, thepigment-producing cells that colonize the epidermis. Melanocytes arederived from the neural crest and migrate during embryologic developmentto selected ectodermal sites (primarily the skin and the CNS) but alsoto the eyes and the ears. Ectopic melanocytes have been identified atautopsy in the gastrointestinal and genitourinary tracts. Congenitalmelanocytic nevi are thought to represent an anomaly in embryogenesisand as such could be considered a malformation or a hamartoma. Incontrast, most acquired melanocytic nevi are considered to be benignneoplastic proliferations.

Atypical moles/dysplastic nevi are acquired melanocytic lesions of theskin whose clinical and histologic definitions are still evolving.Atypical moles differ from common acquired melanocytic nevi in severalrespects, including diameter and lack of pigment uniformity

Birth marks (Capillary hemangiomas) are one of the most common benignorbital tumors of infancy. They are benign endothelial cell neoplasmsthat are typically absent at birth and characteristically have rapidgrowth in infancy with spontaneous involution later in life. This is incontrast to another known group of childhood vascular anomalies,vascular malformations. Vascular malformations, such as lymphangiomasand arteriovenous malformations, are present at birth and arecharacterized by very slow growth with persistence into adult life.

Striae distensae (Stretch marks) affect skin that is subjected tocontinuous and progressive stretching; increased stress is placed on theconnective tissue due to increased size of the various parts of thebody. It occurs on the abdomen and the breasts of pregnant women, on theshoulders of body builders, in adolescents undergoing their growthspurt, and in individuals who are overweight. Skin distension apparentlyleads to excessive mast cell degranulation with subsequent damage ofcollagen and elastin. Prolonged use of oral or topical corticosteroidsor Cushing syndrome (increased adrenal cortical activity) leads to thedevelopment of striae.

Acne manifestation is defined by the distribution of the pilosebaceousglands. Adolescence causes endocrine maturation of the adnexal elements,resulting in an accumulation of cellular products within the ductilesystems. In addition to the cellular products are coexistentmicroorganisms, most commonly Propionibacterium acnes and Staphylococcusepidermidis.

Rosacea is a common condition characterized by symptoms of facialflushing and a spectrum of clinical signs, including erythema,telangiectasia, coarseness of skin, and an inflammatory papulopustulareruption resembling acne. Rosacea is defined by persistent erythema ofthe central portion of the face lasting for at least 3 months.Supporting criteria include flushing, papules, pustules, andtelangiectasias on the convex surfaces. Secondary characteristics areburning and stinging, edema, plaques, a dry appearance, ocularmanifestations, and phymatous changes. Perioral dermatitis (POD) is achronic papulopustular facial dermatitis. It mostly occurs in youngwomen. The clinical and histologic features of the lesions resemblethose of rosacea.

Warts are benign proliferations of skin and mucosa caused by the humanpapilloma virus (HPV). Currently, more than 100 types of HPV have beenidentified. Certain HPV types tend to occur at particular anatomicsites; however, warts of any HPV type may occur at any site. The primaryclinical manifestations of HPV infection include common warts, genitalwarts, flat warts, and deep palmoplantar warts (myrmecia). Less commonmanifestations of HPV infection include focal epithelial hyperplasia(Heck disease), epidermodysplasia verruciformis, and plantar cysts.Warts are transmitted by direct or indirect contact, and predisposingfactors include disruption to the normal epithelial barrier. Treatmentcan be difficult, with frequent failures and recurrences.

Genital warts are a result of human papillomavirus (HPV) infectionacquired by inoculation of the virus into the epidermis via defects inthe epithelium (eg, maceration of the skin). Autoinoculation of virusinto opposed lesions is common. Spread of HPV infection is usuallythrough skin-associated virus and not from blood-borne infection.

Bowenoid papulosis (BP) occur on the genitalia of both sexes in sexuallyactive people. BP is manifested as papules that are induced virally byhuman papillomavirus (HPV) and demonstrate a distinctive histopathology(bowenoid dysplasia).

Psoriasis is characterized by exceedingly rapid turnover of skin andappears as a chronic, bilaterally symmetric, erythematous plaquelikelesion with a silvery scale covering. The lesions classically arelocated over the extensor surfaces, including the elbows, knees, back,and scalp. Confluent generalized lesions also may occur.

In Von Recklinghausen disease multiple neural tumors appear on the body.Numerous pigmented skin lesions occur. The classic café au lait spotspredominate. Additionally, pigmented iris hamartomas (i.e., Lischnodules) are common. Bone lesions and intracranial and GI lesions andsymptoms may be identified.

Necrobiosis lipoidica diabeticorum is a plaquelike, depressed, atrophicyellow lesion typically found in patients with diabetes. It has a strongassociation with diabetes and actually may be a clinical prodrome of theonset of the disease systemically. It rarely is found in locations otherthan the lower extremities and seldom is found in the absence ofdiabetes. The lesion tends to progress from a red plaquelike area to onewith atrophy that occasionally may ulcerate.

Seborrheic dermatitis is a papulosquamous disorder patterned on thesebum-rich areas of the scalp, face, and trunk. In addition to sebum,this dermatitis is linked to Malassezia, immunologic abnormalities, andactivation of complement.

Seborrheic keratosis (also known as seborrheic wart, senile wart, andbasal cell papilloma) is a common benign tumor in advanced andmiddle-aged persons. It is typically a raised papular lesion of variablecolor from light to dark brown. Seborrheic keratosis may be smooth orwartlike with visible pitting. Common sites include the face, trunk, andextremities. The lesion also may be pedunculated or sessile. A variantknown as dermatosis papulosa nigra occurs over the forehead and malarregions of individuals with black skin.

Acrochordons (also known as skin tag, fibroepithelial polyp, fibromamolle, and fibroepithelial papilloma) occasionally are associated withpregnancy, diabetes mellitus, and intestinal polyposis syndromes. Theytend to be located in the intertriginous areas of the axilla, groin, andinframammary regions as well as in the low cervical area along thecollar line. They are soft fleshy papules and usually, although notnecessarily, pedunculated.

Actinic keratosis is the most common sun-related growth. Actinickeratoses are chiefly found on the sun-exposed areas of the face, theears, the forearms, and the dorsum of the hands. However, they may occuron any area that is chronically or repeatedly exposed to the sun, suchas the back, the chest, and the legs. They usually appear as multiplediscrete, flat or elevated, verrucous, keratotic lesions. Lesionstypically have an erythematous base covered by scale (hyperkeratosis).They are usually 3-10 mm in diameter and gradually enlarge into broader,more elevated lesions. With time, actinic keratoses may develop intoinvasive cutaneous horns or skin cancers. Histologically, the epidermalchanges are characterized by acanthosis, parakeratosis, anddyskeratoses. Cellular atypia is present, and the keratinocytes vary insize and shape. Mitotic figures are common.

Bowen disease also is known as carcinoma in situ and squamousintraepidermoid neoplasia. Lesions involve predominantly skin unexposedto the sun (i.e., protected). Classically, Bowen disease involves thegenitalia. Itching is a common complaint. With vulvar involvement, thelabia majora tend to be involved more than the labia minora. The lesionsare scaly, crusted, erythematous plaques.

Pseudocarcinomatous hyperplasia are lesions caused by a reparativeprocess characterized by tongues of squamous epithelium growing downwardinto the dermis.

Nevus sebaceus of Jadassohn is a hamartomatous lesion expressingelements of sebaceous and apocrine glands, defective hair follicles,acanthosis, and papillomatosis. It is a congenital lesion, usuallypresent on the scalp and face. The lesion tends to enlarge with time.

Lupus erythematosus (LE) is a heterogeneous connective-tissue diseaseassociated with polyclonal B-cell activation.

Sebaceous adenoma is a nodular and lobulated lesion with peripheralgenerative cells and variable sebaceous differentiations as the centerof the lesion is approached. It is not as organized as the patterns ofsebaceous hyperplasia. This lesion is distinct from the hamartomatousvariety encountered on the face of patients with tuberous sclerosissyndrome.

Inverted follicular keratosis is believed to be an inflammatory variantof Seborrheic keratosis. It commonly is found on the faces andsun-exposed areas of elderly patients. Typically, this lesion is locatedon the upper eyelid. Anatomically, it represents an upside-down orendophytic process within the epithelium of a pilosebaceous follicle.The lesions tend to be single and present as a papule or nodule.

Trichoepithelioma is an uncommon benign lesion. It is generally pink toflesh colored. It is frequently multiple and is not ulcerative. Theselesions tend to be recapitulations of hair follicles. Initially, theyappear during adolescence. Typical areas for this lesion are the faceand scalp and, less commonly, the trunk and neck.

Trichilemmoma is a benign tumor with a pattern of globular glycogen-richclear cells. Occasionally, keratinization in the center is identifiedgrossly.

Molluscum sebaceum is a self-healing skin tumor. The lesion isclassically a dome-shaped mound with a central crater of keratin.

Basal cell carcinoma is an epithelial malignancy that appears asinsidious, painless, nonhealing ulcers or nodules on the sun-exposedparts of the body. The most common location on the head is the nose,specifically the nasal tip and alae. Risk is related to skin type andthe degree of exposure to sunlight, particularly UV-B radiation. Thetumors are more frequent in individuals with fair complexions.

Most Squamous cell carcinomas appear on sun-exposed regions of the body.Squamous cell carcinoma (SCC) arises from the malignant transformationand proliferation of keratinocytes in the epidermis. SCC can arise fromactinic keratosis, leukoplakia, radiation keratosis or dermatitis,scars, chronic ulcers, or chronic sinusitis. People with actinickeratosis have atypical squamous cells in a third to a half of theepidermis. Those with Bowen disease, or SCC in situ, have atypicalkeratinocytes in the entire epidermis. Invasive SCC involves theepidermis and invades the dermis. The tumors initially appear as skinpatches, plaques, and nodules that enlarge and develop central areas ofinflammation, induration, and, subsequently, necrosis and oozing. SCCsmetastasize by direct, lymphatic, and hematogenous extension.

Melanoma is a tumor that develops as a result of the malignanttransformation of melanocytes. These cells are derived from the neuralcrest. Melanomas usually occur on the skin but can arise in otherlocations where neural crest cells migrate, such as in thegastrointestinal tract or brain.

It is contemplated that the compositions of the present invention finduse in the treatment of all of the foregoing skin conditions anddisorders.

The compositions of the present invention also find use in the treatmentof burns. Sunburn is an acute cutaneous inflammatory reaction thatfollows excessive exposure of the skin to ultraviolet radiation (UVR).Exposure to solar radiation has the beneficial effects of stimulatingthe cutaneous synthesis of vitamin D and providing radiant warmth.Unfortunately, when the skin is subjected to excessive radiation in theultraviolet range (wavelength <400 nm), deleterious effects may occur.The most common is acute sunburn or solar erythema. Eyes, particularlythe cornea (the clear window of tissue on the front of the eyeball), canbe damaged easily by exposure to ultraviolet radiation from the sun andfrom other sources of ultraviolet light, such as a welder's arc, aphotographer's flood lamps, a sun lamp, or even a halogen desk lamp.

Severe burns result in skin barrier destruction that can lead to fluidand electrolyte losses and in skin infection that result in systemicinfection. Burns are rated on the degree of injury to the tissue.First-degree burns involve damage to the top layer of skin (epidermis),and second-degree burns involve the epidermis and the underlying layerof skin (dermis). First- and second-degree burns can also be calledpartial-thickness burns. Third-degree burns affect the epidermis, dermisand hypodermis, causing charring of skin or a translucent white color,with coagulated vessels visible just below the skin surface. These arealso called full-thickness burns.

Treating severely burned patients includes early cleaning and debridingof the wound, intravenous (IV) fluids containing electrolytes, systemicantibiotics, topical antibiotics, nutritional support and medication tocontrol pain. Skin grafting, generally with skin taken from donor sitesfrom the patient, may be required to achieve closure of the woundedarea. In large burns, autograft skin may not be available in sufficientquantities to completely close the wound. In this case, expandedautografts are applied to the wound, and cadaver allograft is then usedto close the wound completely. Skin graft donor sites are surgicallycreated wounds that require the same level of care as other open wounds.

The compositions of the present invention also find use in the treatmentof various types if internal wounds. Wounds on internal tissues may bethe result disease of surgery such as those created by of removal ofcancerous tissues or correction of a cleft lip and/or palate. Wounds canform on the membranes of the mouth, nose and digestive system.

A cleft lip or palate affects the obvious facial form as an anatomicdeformity and has functional consequences, affecting the child's abilityto eat, speak, hear, and breathe. Specifically, in the child born with abilateral cleft, the surgeon initially is faced with a protrusivepremaxilla and the difficulty of achieving adequate columellar lengthand vertical height to the lip during reconstruction. Although surgeryfor the bilateral cleft lip has undergone many recent advances,correction of the nasal deformities associated with this congenitalmalformation remains one of the greatest challenges in plastic surgery.Surgical correction of nasal deformities associated with bilateral cleftlip is challenging because deformities may become apparent as the noseundergoes further growth and development.

Removal of cancer from the jawbone often creates a gap in the bone thatwounds surrounding tissues. Distraction osteogenesis is a technique inwhich bone can be lengthened by de novo bone formation as part of thenormal healing process that occurs between surgically osteotomized bonesegments that undergo gradual, controlled distraction.

Velopharyngeal (VP) dysfunction includes any structural and/orneuromuscular disorder of the velum and/or pharyngeal walls at the levelof the nasopharynx in which interference with normal sphincteric closureoccurs. VP dysfunction may result from anatomic, myoneural, behavioral,or a combination of disorders

Erythema multiforme (EM) is an acute mucocutaneous hypersensitivityreaction of variable severity characterized by a symmetricallydistributed skin eruption, with or without mucous membrane lesions. Themore common mild form, EM minor, consists of skin lesions withinvolvement of no more than one mucosal surface. Symmetricallydistributed, erythematous, expanding macules or papules evolve intoclassic iris or target lesions, with bright red borders and centralpetechiae, vesicles, or purpura. EM major, or Stevens-Johnson syndrome,is more severe, involving 2 or more mucous membranes with more variableskin involvement. It may involve internal organs and typically isassociated with systemic symptoms. Skin findings may be similar to EMminor but often are more variable and severe. Inflammatoryvesiculobullous lesions, often with hemorrhage and necrosis, aretypical.

Rhinitis is defined as inflammation of the nasal membranes and ischaracterized by a symptom complex that consists of any combination ofthe following: sneezing, nasal congestion, nasal itching, andrhinorrhea. The eyes, ears, sinuses, and throat can also be involved.Allergic rhinitis is the most common cause of rhinitis.

Crohn disease is an idiopathic, chronic, transmural inflammatory processof the bowel that can affect any part of the GI tract from the mouth tothe anus. The condition is believed to be the result of an imbalancebetween proinflammatory and anti-inflammatory mediators. Most casesinvolve the small bowel, particularly the terminal ileum. Thecharacteristic presentation of Crohn disease is with abdominal pain anddiarrhea, which may be complicated by intestinal fistulization,obstruction, or both. The initial lesion starts as a focal inflammatoryinfiltrate around the crypts, followed by ulceration of superficialmucosa. Later, inflammatory cells invade deep layers and, in thatprocess, begin to organize into noncaseating granulomas. The granulomasextend through all layers of the intestinal wall and into the mesenteryand the regional lymph nodes. Although granuloma formation ispathognomonic of Crohn disease, absence does not exclude the diagnosis.The initial abnormality is hyperemia and edema of the involved mucosa.Later, discrete superficial ulcers form, which become deep serpiginousulcers located transversely and longitudinally over an inflamed mucosa,giving the mucosa a cobblestone appearance. The lesions are oftensegmental, being separated by healthy areas. Malabsorption occurs asresult of loss of functional mucosal absorptive surface. This phenomenoncan lead to protein-calorie malnutrition, dehydration, and multiplenutrient deficiencies. Involvement of the terminal ileum may result inmalabsorption of bile acids, which leads to steatorrhea, fat-solublevitamin deficiency, and gallstone formation. Fat malabsorption, bytrapping calcium, may result in increased oxalate excretion (normallycomplexed by calcium), causing kidney stone formation.

Gastritis includes a myriad of disorders that involve inflammatorychanges in the gastric mucosa, including erosive gastritis caused by anoxious irritant, reflux gastritis from exposure to bile and pancreaticfluids, hemorrhagic gastritis, infectious gastritis, and gastric mucosalatrophy. Peptic ulcer disease (PUD) refers to a discrete mucosal defectin the portions of the gastrointestinal tract (gastric or duodenal)exposed to acid and pepsin secretion. Erosive gastritis usually isassociated with serious illness or with various drugs. Stress, ethanol,bile, and nonsteroidal anti-inflammatory drugs (NSAIDs) disrupt thegastric mucosal barrier, making it vulnerable to normal gastricsecretions. Infection with Helicobacter pylori, a short, spiral-shaped,microaerophilic gram-negative bacillus, is the leading cause of PUD andis associated with virtually all ulcers not induced by NSAIDs.

Oral herpes is an infection caused by the herpes simplex virus. Thevirus causes painful sores on your lips, gums, tongue, roof of yourmouth, and inside your cheeks. It also can cause symptoms such as feverand muscle aches.

The compositions of the present invention further find use in enhancingthe various phases of the healing process. There are different phases tothe healing process. The inflammatory phase begins with the injuryitself. The inflammatory phase is characterized by hemostasis andinflammation. Here you have bleeding, immediate narrowing of the bloodvessels, clot formation, and release of various chemical substances intothe wound that will begin the healing process. Specialized cells clearthe wound of debris over the course of several days. Collagen exposedduring wound formation activates the clotting cascade (both theintrinsic and extrinsic pathways), initiating the inflammatory phase.After injury to tissue occurs, the cell membranes, damaged from thewound formation, release thromboxane A2 and prostaglandin 2-alpha,potent vasoconstrictors. This initial response helps to limithemorrhage. After a short period, capillary vasodilatation occurssecondary to local histamine release, and the cells of inflammation areable to migrate to the wound bed.

Platelets, the first response cell, release multiple chemokines,including epidermal growth factor (EGF), fibronectin, fibrinogen,histamine, platelet-derived growth factor (PDGF), serotonin, and vonWillebrand's factor. These factors help stabilize the wound through clotformation. These mediators act to control bleeding and limit the extentof injury. Platelet degranulation also activates the complement cascade,specifically C5a, which is a potent chemoattractant for neutrophils. Theinflammatory phase continues, and more immune response cells migrate tothe wound. The second response cell to migrate to the wound, theneutrophil, is responsible for debris scavenging, complement-mediatedopsonization of bacteria, and bacteria destruction via oxidative burstmechanisms (i.e., superoxide and hydrogen peroxide formation). Theneutrophils kill bacteria and decontaminate the wound from foreigndebris. The next cells present in the wound are the leukocytes and themacrophages (monocytes). The macrophage, referred to as theorchestrator, is essential for wound healing. Numerous enzymes andcytokines are secreted by the macrophage. These include collagenases,which debride the wound; interleukins and tumor necrosis factor (TNF),which stimulate fibroblasts (produce collagen) and promote angiogenesis;and transforming growth factor (TGF), which stimulates keratinocytes.This step marks the transition into the process of tissuereconstruction, i.e., the proliferative phase.

In the proliferative phase a matrix or latticework of cells forms. Onthis matrix, new skin cells and blood vessels will form. It is the newsmall blood vessels (known as capillaries) that give a healing wound itspink or purple-red appearance. These new blood vessels will supply therebuilding cells with oxygen and nutrients to sustain the growth of thenew cells and support the production of proteins (primarily collagen).The collagen acts as the framework upon which the new tissues build.Collagen is the dominant substance in the final scar.

Epithelialization, angiogenesis, granulation tissue formation, andcollagen deposition are involved in the proliferation phase anabolicportion of wound healing. Epithelialization occurs early in woundrepair. If the basement membrane remains intact, the epithelial cellsmigrate upwards in the normal pattern. This is equivalent to afirst-degree skin burn. The epithelial progenitor cells remain intactbelow the wound, and the normal layers of epidermis are restored in 2-3days. If the basement membrane has been destroyed, similar to a second-or third-degree burn, then the wound is reepithelialized from the normalcells in the periphery and from the skin appendages, if intact (eg, hairfollicles, sweat glands)

Angiogenesis, stimulated by TNF-alpha, is marked by endothelial cellmigration and capillary formation. The new capillaries deliver nutrientsto the wound and help maintain the granulation tissue bed. The migrationof capillaries into the wound bed is critical for proper wound healing.The granulation phase and tissue deposition require nutrients suppliedby the capillaries, and failure for this to occur results in achronically unhealed wound. Mechanisms for modifying angiogenesis areunder study and have significant potential to improve the healingprocess.

During granulation tissue formation, fibroblasts differentiate andproduce ground substance and then collagen. The ground substance isdeposited into the wound bed; collagen is then deposited as the woundundergoes the final phase of repair. Many different cytokines areinvolved in the proliferative phase of wound repair. The steps and theexact mechanism of control are not well understood. Some of thecytokines include PDGF, insulin-like growth factor (IGF), and EGF.

During a remodeling stage, the framework (collagen) becomes moreorganized making the tissue stronger. The blood vessel density becomesless, and the wound begins to lose its pinkish color. Over the course of6 months, the area increases in strength, eventually reaching 70% of thestrength of uninjured skin. In the maturational phase, the woundundergoes contraction, ultimately resulting in a smaller amount ofapparent scar tissue. The entire process is a dynamic continuum with anoverlap of each phase and continued remodeling. The wound reachesmaximal strength at one year, with a tensile strength that is 30% ofnormal skin. Collagen deposition continues for a prolonged period, butthe net increase in collagen deposition plateaus after 21 days.

Epithelialization is the process of laying down new skin, or epithelial,cells. The skin forms a protective barrier between the outer environmentand the body. Its primary purpose is to protect against excessive waterloss and bacteria. Reconstruction of this layer begins within a fewhours of the injury and is complete within 24-48 hours in a clean,sutured (stitched) wound. Open wounds may take 7-10 days because theinflammatory process is prolonged, which contributes to scarring.Scarring occurs when the injury extends beyond the deep layer of theskin (into the dermis).

The 3 categories of wound closure are primary, secondary, and tertiary.Primary healing involves closure of a wound within hours of itscreation. Secondary healing involves no formal wound closure; the woundcloses spontaneously by contraction and reepithelialization. Tertiarywound closure, also known as delayed primary closure, and involvesinitial debridement of the wound for an extended period and then formalclosure with suturing or by another mechanism.

The compositions of the present invention further find use for thetreatment of scars, alone or in combination with known scar treatments.Open wounds can result in a number of complications including woundinfection and disfiguring scars including keloids, widened scars, andhypertrophied scars. Both keloid and hypertrophic scars are wounds thatheal overzealously above the skin surface. The difference between akeloid and a hypertrophied scar is that a keloid continues to enlargebeyond the original size and shape of the wound, while a hypertrophiedscar enlarges within the confines of the original wound. Although bothcan be red and raised, keloids continue to grow and hypertrophied scarstend to regress over time. Both can recur after surgical excision;however, the recurrence of keloid scars is more common. Widened scarsare wounds that separate during the healing process, usually in responseto tension perpendicular to the wound edges. Hypertrophic scars are morecommon than keloids. Hypertrophic scars may occur in persons of any ageor at any site, and they tend to spontaneously regress. In general,hypertrophic scars are more responsive to treatment. While keloids occurfrequently in black persons, they may occur in persons of any race witha proven tendency to keloid formation. Keloids are more prevalent inpersons aged 10-30 years, while hypertrophic scars occur in persons ofany age.

Which factors initiate keloid or hypertrophic scar formation is not wellunderstood. Several genetic and environmental causes have beenimplicated in the etiology of keloid and hypertrophic scars. In bothkeloid and hypertrophic scar formation, an excessive accumulation ofcollagen from increased collagen synthesis or decreased collagendegradation occurs. Proposed causes for abnormal scar formation includeforeign body reaction and bacterial infections. Many abnormal scars areassociated with tattoos, burns, injections, bites, vaccinations, trauma,surgery, or infection. Skin tension is frequently implicated inhypertrophic scar formation. Abnormal scar healing commonly involvesareas of high skin tension, such as the anterior chest, shoulders, andupper back. Other factors implicated in the etiology of abnormal scarformation include wound infection or anoxia, a prolonged inflammatoryresponse, and wound orientation different from the relaxed skin tensionlines. Keloid formation has a genetic basis, as demonstrated by itspredilection for persons of certain races and in certain families.Because keloids tend to demonstrate accelerated growth during puberty orpregnancy and tend to resolve with menopause, hormones (both androgenand estrogen) have been implicated in keloid formation. Other hormoneslinked to keloid formation include thyroid hormone alterations andmelanocyte-stimulating hormones. Immunologic alterations are implicatedin abnormal scars. Specifically, irregular immunoglobulin and complementlevels, increased transforming growth factor-beta, and mast cells arefound in abnormal scars. Additionally, decreased tumor necrosis factorand interleukin 1 levels are found in these abnormal scars. Widenedscars result from excess tension perpendicular to the wound edges duringthe healing process. Scar widening usually occurs within the first 6months of injury.

Although multiple factors are involved in abnormal scar formation,studies indicate that keloid and hypertrophied scars result fromincreased collagen production and decreased collagen degradation. Levelsof the collagen-related enzyme prolyl hydroxylase are elevated inkeloid-affected skin compared with normal skin. Prolyl hydroxylase isrequired for the hydroxylation of proline during collagen synthesis,suggesting that collagen overproduction occurs with keloids.

Collagen production is elevated in keloid biopsy samples and in culturedfibroblasts derived from keloids. Increased collagen production bycultured fibroblasts derived from keloids persists throughout their invitro life span; they do not revert to normal after transfer of thelesion to culture. No significant differences in DNA content orcellularity are found when keloid dermis is compared with normal dermis.This suggests that each fibroblast is producing more collagen ratherthan an increase occurring in the number of fibroblasts producing anormal amount of collagen. In keloid formation, excessive collagenproduction by fibroblasts is likely due to the wound environment.

Widened scar formation is thought to result from wound edge separationwith tension perpendicular to the healing skin wound. A state of tensionexists naturally in skin; wounded skin gapes and becomes ellipticalrather than round. When a wound is closed opposite to the lines oftension, the chance of widened scar formation is increased.

Upon clinical examination, keloids and hypertrophic scars are raisedabove the skin level. Hypertrophic scars are self-limited; theyhypertrophy within the confines of the wound. Initially, hypertrophiedscars can be raised, red, pruritic, and even painful; however, overtime, they become pale and flat. Hypertrophied scars appear worst at 2weeks to 2 months. Keloid scars can be differentiated from hypertrophicscars by their spread beyond the original wound. Keloid scars tend toremain red, pruritic, and painful for many months to years untilmenopause. Patients usually have a personal or familial history ofkeloid formation. Different from hypertrophic and keloid scars, widenedscars are flat and sometimes depressed. With adequate wound maturation,these wounds fade to the pigment of the surrounding uninjured skin.Widened scars are not usually red or pruritic.

The relaxed skin tension lines follow furrows formed when the skin isrelaxed. Unlike wrinkles, they are not visible features of the skin.They are merely derived from the furrows produced by pinching on theskin. These furrows are produced preferably with pinching perpendicularto the lines. When the skin is pinched oblique to the relaxed skintension lines, an S-shaped pattern is created. Fewer and higher furrowsare created if skin is pinched parallel to the lines. Closing incisionsopposite to the relaxed skin tension lines can increase the risk ofwidened or hypertrophic scar formation.

A potential relative contraindication to scar revision surgery existswhen the scar is a keloid because of the risk of worsening the scar.Sometimes, when keloids recur, they become larger than the original.Widened scars can be easily differentiated from hypertrophic and keloidscars based on findings from a physical examination. Widened scars areflat and sometimes even depressed. Hypertrophic scars and keloids areindistinguishable under light microscopy. However, there are a number ofdifferences when viewed under an electron microscope and when evaluatedimmunochemically. Keloids contain thick collagen fibers with increasedepidermal hyaluronic content, whereas hypertrophic scars exhibit nodularstructures with fine collagen fibers and increased levels of alphasmooth muscle actin. The collagen in both keloids and hypertrophic scarsis organized in discrete nodules, frequently obliterating the rete pegsin the papillary dermis of the lesions. While collagen in normal dermisis arranged in discrete fascicles separated by considerable interstitialspace, collagen nodules in keloids and in hypertrophic scars appearavascular and unidirectional and are aligned in a highly stressedconfiguration.

Different nonsurgical options treat abnormal scars. Pressure is thoughtto decrease tissue metabolism and increase collagen breakdown within thewound. The different methods of applying pressure include the use ofelastic bandages (ACE wraps), thromboembolic disease stockings, orIsotoner-type gloves on extremities. Alternatively, custom-fittedcompression garments can be used to apply pressure to the more difficultareas, including the neck and torso. Because these devices areuncomfortable, patient compliance varies. Unfortunately, for optimalresults, these devices must be used for 6-12 months during thematuration of the wound.

Silicone gel can be used to treat abnormal scars. Silicone gel is shownto significantly decrease scar volume when used over time particularlyfor hypertrophic scar formation. The effect of the silicone gel on thescar is believed to be due to wound hydration. The silicone gel isapplied to the wound for at least 12 h/d. Patients find it moreappealing to apply the silicone to their wounds at night. Silicone gelis gaining popularity because it can be applied to a smaller area for 12h/d, usually at night. However, skin breakdown, rashes, and difficultywith wound adherence can lead to disuse.

Steroid injections have become a common nonsurgical option in thetreatment of problem scars. The steroid used for intralesional injectionis triamcinolone (Kenalog). Triamcinolone injections have been thestandard treatment to induce flattening, fading, and decreasedsymptomatology of hypertrophied scars. These injections can beadministered as soon as a problem scar is identified. The dose of theinjection can vary from 10-120 mg, depending on the size of the scar.

One may make use of a triamcinolone injection for thin-to-widehypertrophied scars and silicone for very wide hypertrophied scars. Somepatients prefer triamcinolone injections to avoid applying and wearingthe silicone every day for 6-9 months, especially on body areas whereadherence is poor. Adverse effects of triamcinolone injections includehypopigmentation and subcutaneous atrophy. Other nonsurgical optionsinclude corticosteroid intralesional injections, vitamin E therapy, zincoxide therapy, antineoplastic agents, and immunotherapy.

If nonoperative measures are unsuccessful in the treatment of abnormalscars, operative intervention can be considered. Closing wounds toorient the wound along the relaxed skin tension lines is important. Astandard practice often used rather subconsciously after excision of alesion involves assessing the direction of least tension based on theconfiguration of the edges of the wound or by pinching the wound.

The first-line procedure used for scar revision is fusiform excision. Ingeneral, fusiform excision does not require lengthening the scar. Inorder to avoid canine auricles, ensure the wound has a length-to-widthratio of 4:1. Fusiform excision is preferred for short wounds orientedalong relaxed skin tension lines. The Millard flap procedure is similarto fusiform excision, but it involves preserving the scar and itsconnection to the underlying fat. The skin is incised in a fusiformfashion around the scar to the subcutaneous level. The scar is thendeepithelialized, and the skin edges are approximated over thedeepithelialized scar. The Millard flap technique is preferred forwidened, depressed scars.

Scars not oriented along the relaxed skin tension lines can be modifiedwith a Z-plasty procedure. Limbs of equal length are created for the Zplasty. The angle of the Z dictates the length of scar tensiondistribution and elongation (eg, 30° for 25%, 45° for 50%, 60° for 75%,75° for 100%, 90° for 120%). The W-plasty technique for scar revision issimilar to Z plasty because of the result of breaking up a straight-linescar into a pattern that is less conspicuous. Similar to a fusiformexcision, W plasty involves the removal of skin; therefore, avoid thismethod if significant tension is present across the wound edges.W-plasty scar revision is preferred for scars along relaxed skin tensionlines; scars with a bowstring contracture; short, depressed scars; andfacial scars.

Tissue expansion and serial excision can be considered for larger scarrevisions when excess wound tension is predicted. If more than 2 serialexcisions are expected, tissue expansion is preferred. Finally, otherprocedures that have been described to treat scars include dermabrasion,cryosurgery, and laser therapy. Widened scars may be treated differentlythan hypertrophied scars. Widened scars can be flat or even depressed.Therefore, the administration of intralesional steroids is notpreferred; these agents might worsen the depression. Widened scars arepreferably treated with the Millard 2-flap technique over adeepithelialized scar. This technique provides soft tissue fill underthe approximated wound edges. Furthermore, if the widened scar recurs,the risk for another recurrence may be minimized by reorienting thewound tension along the lines of relaxed skin tension. Other adjunctsdescribed in the treatment of widened scars include the injection of fatgrafts or other tissue substitutes. When oriented close to the relaxedskin tension lines, hypertrophic scars can be excised in a fusiformfashion. If the hypertrophic scar developed because of excessive tensionacross the wound as a result of unfavorable wound orientation, Z plastycan sometimes help reorient the wound to distribute tension in adifferent direction to minimize the risk of recurrence.

Postoperatively, compression garments and silicone gel are preferred for4-6 months to decrease the risk of recurrence. Patients are encouragedto refrain from strenuous activities for at least 6 weeks, until whichtime the wound achieves approximately 80% original wound tensilestrength. Patients are monitored for 6 months postoperatively to detectand potentially circumvent recurrences early. Postoperatively, patientsare at risk for hypertrophic scar and widened scar recurrence. Otherrisks include infection, hematoma, seroma, and painful or unattractivescarring. The risk of recurrence is significant for both hypertrophicand widened scars, and it is increased with repeat operations. Woundhealing requires approximately 1 year, during which time the surgeon andpatient should observe for and expect improvement. Once the scar has hadan opportunity to mature, scar revision can be considered.

In some embodiments of the present invention, compositions comprisingdifferentiable cell extracts are utilized to improve any area of theperson visible and contributing to cosmetic appearance of a person,including but not limited to skin, hair, nails, teeth, subcutaneous fat,cartilage, muscle and skeletal structures. The described gene-gun andmicroinjection delivery methods are contemplated to introduce extractsor extract components to structures below the surface skin of a person.

This invention relates to prevention of deterioration, damage andmalfunction of cells and tissues, and to promote, improve and exceedcellular function in order to promote, improve and exceed appearance,vitality and health by treating cells and tissues with differentiablecells, cell or egg extracts, or components of said extracts includingsignaling molecules, peptides, carbohydrates, lipids or nucleic acids.

The current invention contemplates the assessment of a person's needsfor healing, regeneration or repair of damage by several means,including but not limited to analysis and measurements of visiblesurfaces, skin pH, thickness, structure and elasticity of skin layers,analysis of blood or tissue samples by microchip, RT-PCR, Massspectrometry, high pressure liquid chromatography, ELISA-assays, RNAanalysis, analysis of accumulation of DNA damage or defective genes byDNA sequencing, assessment of internal organ and tissue health by X-rayimaging, ultrasound imaging, computed tomography (CT), magneticresonance imaging (MRI), positron emission tomography (PET).

Subcutaneous fat contributes to the cosmetic appearance of a person, andis redistributed during ageing, by smoking and in a number of diseases,including HIV and diabetes as well as in burn-victims. The humanimmunodeficiency virus (HIV)-lipodystrophy syndrome is associated withfat redistribution and metabolic abnormalities, including insulinresistance. Increased intramyocellular lipid (IMCL) concentrations arethought to contribute to insulin resistance, being linked to metabolicand body composition variables. Among HIV-infected subjects, calfsubcutaneous fat area and extremity fat are reduced. Extremity fat issignificantly associated with IMCL among HIV-infected patients,controlling for visceral abdominal fat, abdominal subcutaneous fat, andantiretroviral medications in a regression model. Increased IMCL inHIV-infected women with a mixed lipodystrophy pattern are mostsignificantly associated with reduced extremity fat. (Torriani M et al.,J. Appl. Physiol. 2006 February; 100(2):609-14. Epub 2005 Oct. 13).Saturation of the subcutaneous fat depot is the primary event in thepathophysiology of insulin resistance in the majority of patients andpostulate that this seminal event may lead to the development ofhypertension, hypertriglyceridemia and depressed HDL levels (i.e., themetabolic syndrome). There are no current effective means toredistribute subcutaneous fat in such persons, current treatment include(1) weight loss with differing responses seen with regards to insulinresistance depending on the size of the fat depot; (2) peroxisomeproliferator activated receptor gamma agonists, such asthiazoledinediones which expand the subcutaneous fat depot, (3)expanding alternate storage sites for triglycerides by a variety oftechniques, such as resistance training-induced muscle hypertrophy, mayalso improve insulin resistance; (4) drugs, such as beta 3 adrenergicreceptor agonists which promote lipolysis may increase insulinresistance by releasing free fatty acids into the circulation.Inhibitors of the beta oxidation of free fatty acids (e.g., carnitinepalmitoyl transferase inhibitors) may cause insulin resistance bysparing fat and (5) liposuction, by reducing the size of thesubcutaneous fat depot may worsen insulin resistance, thus increasingthe risk of type 2 diabetes mellitus (Cherian M A, Santoro T J, MedHypotheses. 2005 Dec. 14; [Epub ahead of print]).

Alterations in subcutaneous fat and skin condition due to hormonechanges that occur during ageing and disease are also contemplated areasof use for this invention. Effects of ovarian and other steroids areimportant to the metabolism of skin and hair, the changes in bodycomposition and the alterations of the subcutaneous fat distributionthroughout life. So called aesthetic endocrinology accesses deficiencyor excess of ovarian steroids that lead to different problems skin andhair and other non-genital, i.e., obesity and cellulite. Sex steroidsare small molecules that are transported into the skin by topicalapplication when properly formulated, and are contemplated to be addedto the extracts presented in this invention in order to achieve localeffects but to avoid systemic reactions. Estrogens, delivered orally ortopically, may counteract the aging of the skin especiallypost-menopause. Estrogen alone is not sufficient for reconstitution ofjuvenile skin but may slow the skin aging process. The hitherto onlysuccessful treatment of hair loss in women is by application of thenon-hormonal compound minoxidil, and compositions contemplated by thisinvention may serve to be a different way of treating hair loss. Indeed,the compositions contemplated may stimulate hair sack follicles toregrow or increase the rate and quality of hair, as well as nails.Estrogens also contribute to hirsutism (the excessive growth of thickdark hair in locations where hair growth in women usually is minimal orabsent), acne and changes in body composition. (Gruber C J, et al.,Current concepts in aesthetic endocrinology. Gynecol Endocrinol. 2002December; 16(6):431-41). The compositions in the present invention areadditionally contemplated for use in hair loss and baldness in maleswhich may be caused by hormones, diet, cancer, chronic illness orstress.

It is contemplated that the present invention can be used to regulatehair growth by stimulating or modulating hair follicle cells to eitherreduce or enhance or regenerate hair growth in desired areas by topicalor sub-dermal applications.

This invention is also useful in the treatment of cellulite. Celluliteis a common term used to describe superficial pockets of trapped fat,which cause uneven dimpling or “orange peel” skin. It appears in 90% ofpost-adolescent women and is rarely seen in men. Common but notexclusive areas where cellulite is found, are the thighs, buttocks, andthe abdomen. Contrary to popular belief, cellulite is not related toobesity, since it occurs in overweight, normal, and thin women.Cellulite can be aided by mechanized devices with motorized rollers andregulated suction. This non-surgical and non-invasive device creates asymmetrical skin fold, which allows for deep tissue mobilization tooccur and results in reduction of cellulite and loss of inches. Thepresent invention contemplates application of extracts topically orsubcutaneously to regulate the distribution of subcutaneous fat depositsand improve the cosmetic appearance of areas affected by cellulite.

It is contemplated that the present invention may be useful for therepair or rejuvenation or de novo formation of damaged tissues, organsand cells beneath the skin, including all internal organs and tissues,including but not limited to muscle, fat, cartilage, bone, connectivetissue, spleen, liver, pancreas, lungs and nervous tissue. Damages tothe internal tissues or organs may be induced by i.e. accidents,diseases, medication, cancer, radiation and surgery.

When the body is exposed to high doses of radiation, a complexbiological response is initiated that may lead to multi-organ failure(MOF). MOF begins with energy deposits in cellular targets and ispropagated and amplified by the tissue response to cell damage. Thebiology of wound healing is at the root of MOF following surgicaltrauma, inflammation is the basis for MOF in sepsis, and the biology ofthe irradiated tissue initiates radiogenic MOF. Tissue response toradiation damage has been suggested to be initiated and coordinated byextracellular signaling. It has been demonstrated that transforminggrowth factor-β1 orchestrates the biology of irradiated tissue as atissue level sensor of oxidative stress, and is integral to the cellularDNA damage response. (Barcellos-Hoff M H. How tissues respond to damageat the cellular level: orchestration by transforming growth factor-β(TGF-β) British Journal of Radiology (2005) Supplement 27, 123-127).

In some embodiments, the compositions described above are used toincrease collagen production by skin cells. In some embodiments, thecompositions are applied to the skin or wounds in the skin in aneffective amount, which is the amount required to increase collagenproduction in the cells. It is contemplated that by increasing collagenproduction, the compositions of the present invention enhance or improvewound healing in a subject. It is also contemplated that by increasingcollagen production upon topical application, the compositions of thepresent invention can improve attributes of damaged skin, such asgeneral appearance, suppleness, smoothness, amount of wrinkles,moisture, color, etc. Accordingly, the composition of the presentinvention find use in increasing the collagen content in skin that hasbeen contacted by the composition so that skin moisture is improved orincreased, skin wrinkling is improved or decreased, skin suppleness isimproved or increased, skin smoothness is improved or increased, skintone is improved or increased, skin color is improved or normalized,skin stretch marks are improved, decreased, or eliminated or skinroughness is improved or decreased. In other embodiments, thecompositions of the present invention are useful for the prophylaxis orprevention of the foregoing skin conditions.

In some embodiments, the compositions described above are used toincrease the proliferation of skin cells, and in particular skinfibroblasts. In some embodiments, the compositions are applied to theskin or wounds in the skin in an effective amount, which is the amountrequired to increase fibroblast proliferation at the site ofapplication. It is contemplated that by increasing fibroblastproliferation, the compositions of the present invention enhance orimprove wound healing in a subject. It is also contemplated that byincreasing fibroblast proliferation upon topical application, thecompositions of the present invention can improve attributes of damagedskin, such as general appearance, suppleness, smoothness, amount ofwrinkles, moisture, color, etc. Accordingly, the composition of thepresent invention find use in increasing the collagen content in skinthat has been contacted by the composition so that skin moisture isimproved or increased, skin wrinkling is improved or decreased, skinsuppleness is improved or increased, skin smoothness is improved orincreased, skin tone is improved or increased, skin color is improved ornormalized, skin stretch marks are improved, decreased, or eliminated orskin roughness is improved or decreased.

Accordingly, in some embodiments, the present invention provides methodsof treating subjects suffering from one or more of the conditionsdescribed above. In some embodiments, the methods comprise contactingthe subject with a composition comprising a differentiable cell extractas described above, or any of the compositions described in detailabove. In some embodiments, the methods comprise contacting the tissueof the subject with an egg extract under conditions such that expressionof a gene is increased, wherein said gene is selected from the groupconsisting of collagen 1, collagen 3, VEGF-B, VEGF-C, TGFβ2, TGFβ3,PDGF-A, PDGF-B, PDGF-D, IL-18, and fibronectin. In some embodiments, themethods comprise contacting the tissue of the subject with an eggextract under conditions such that expression of a gene is decreased,wherein said gene is selected from the group consisting of a matrixmetallopeptidase, TGFβ1, VEGF-A, elastin, IL 1β, and IL 12. In someembodiments, the matrix metallopeptidase (MMP) is selected from thegroup consisting of MMP 14, 16, 17, 19, 20, 23, 25 and 28. In someembodiments, the present invention provides methods of treating asubject with skin condition comprising contacting the skin of saidsubject with a fish or amphibian cytoplasmic egg extract in an effectiveamount, wherein said skin condition is selected from the groupconsisting of ulcers, psoriasis, calluses, moles, acne, rosacea,dermatitis, keratosis, basal cell carcinoma and squamous cell carcinoma.In preferred embodiments, the differentiable cell extract is a fish oramphibian cytoplasmic cell extract and is provided in a cream, gel,emulsion, ointment, spray, powder or lotion. In some embodiments, thepresent invention provides compositions comprising a differentiable cellextract for use in treating or preventing any of the conditionsdisclosed above.

M. Whole Cell Applications

In some embodiments of the present invention, compositions comprisingintact stem cells (embryonic or adult) or cord-blood stem cells areutilized for cosmetic or therapeutic purposes. In some embodiments,suspensions of cells in fluid form are introduced to the skin. In someembodiments, suspensions of cells in fluid form are introduced into anopen wound, and then covered by a wound dressing which can breathe (nonocclusive). In some embodiments, an occlusive wound dressing isutilized. In some embodiments, one or more layers are utilized, forexample a waterproof plastic membrane which can be glued onto skin, alayer of a nutrient gel which can nourish cells and speed wound healing(containing antibacterial agents, collagen modulating substances andother substances); and a layer of skin stem cells embedded in/placed onthe nutrient layer, which are put in direct contact with the wound. Insome embodiments, the cells are cultured in the lab from the person'sown skin, adipose, or stem cells. In some preferred embodiments, thecells are then harvested and put in suspension, either to be applied asa fluid or placed on a plastic membrane with nutrient gel-layer to beapplied to skin as an occlusive wound dressing/plaster/band-aid.

N. Ex Vivo and In Vivo Therapy

In some embodiments, the extracts are utilized for ex vivo treatment ofcells derived from a patient. Briefly, cells are recovered from apatient, expanded, permeabilized, incubated with the extract, sealed,and then used for treatment of a patient. In this process, a number ofthe cells properties could be altered or enhanced, including but notlimited to lengthening of telomeres—the terminal chromosomes protectingthe central DNA contained in the chromosome which are shortened witheach cell division—thus renewing and lengthening the life-span of thecell treated. Preferred methods are described in Example 3.

In some embodiments, the extracts are utilized in vivo on the patient'sinternal organs and/or tissues or cells. Briefly, the extract orcomponents thereof could be injected to the intraperitoneal cavity, thusbathing the surface of abdominal organs including but not limited to theintestines, liver, spleen, pancreas, stomach and bladder thus inducinghealing of wounds in these organs and tissues or aiding in regenerationof the cells which the organs and/or tissues are composed of.

It is also contemplated to introduce cells or extracts or componentsthereof into internal organs and/or tissues including but not limited tomuscle, brain, fat, connective tissue, cartilage, pancreas, liver,spleen, heart and lungs as to induce de novo cell formation in tissuesand organs and/or rejuvenate the cells from which the tissues/organs arecomposed. De novo cell formation occurs spontaneously in organismsincluding humans. De novo formation of local lymphoid tissue bydendritic dells which are the most potent professionalantigen-presenting cells (Ludewig B et al., 1998 J Exp Med).

Life is manifested in growth. In plants, growth can be of two types,heterotrophic and autotrophic. Autotrophic growth uses inorganicmaterial for nourishment. Heterotrophic growth is dependent on organicmaterial for nourishment. During germination, seedlings usually growheterotropically but once a plant becomes photosynthetic it can growautotrophically—using minerals from the soil and atmosphere and sunlightfor energy. Thus, for most of their life plants are autotrophic.However, there are some parasitic plants that grow heterotropically,obtaining inorganic material from their host. In plants, growth isserial, repetitive and plastic, and cell divisions contribute to de novoformation of organs all the way through to senescence. In animals, celldivision serves to regenerate and maintain tissues and circulating cellpopulations, and growth is concurrently repetitive and dependent on thelength of the telomeres which are shortened at each cell division.

This invention contemplates using both organic materials and inorganicmaterials contained in or added to extracts to nourish, stimulate andregulate cell growth, function and de novo formation in all organs andtissues. Plant seeds contain materials for heterotropic growth and arecontemplated for use in extracts to aid cell growth in humans.

Certain animals have the ability to regenerate parts of their bodiesafter loss or injury. To actually regrow a lost organ or other structurerather than simply fill the void with scar tissue involves processesranging from an injury response and wound healing to growth, patterningand differentiation of new tissues similar to that which occurred duringembryonic development. With new evidence for the presence of stem cellsin most if not all adult organs and their ability to participate intissue repair, the field of regenerative biology has assumed much morewidespread medical relevance (Stocum, 1995, 2004). If one looks at thephylogenetic distribution of regenerative ability in various organsystems, it appears that this capacity has been lost gradually in thecourse of animal evolution (Thouveny and Tassava, 1998; SanchezAlvarado, 2000). Regeneration of amputated limbs in amphibians is one ofthe best-studied model systems and a useful paradigm for understandingmany features of vertebrate organ regeneration. As an example of“epimorphic” regeneration, this system includes cellulardedifferentiation in the injured tissues of the limb stump andproliferation of these cells to form a distal blastema that undergoespatterning and growth to restore the missing limb structures. Thequestion of why limbs of phylogenetically advanced vertebrates fail toregenerate has been addressed by studies with limbs of anuranamphibians. Regeneration is excellent in the earliest stages of limbdevelopment in anurans (frogs and toads) but gradually diminishes aslarvae approach metamorphosis (Dent, 1962). Limbs of adult anurans areincapable of complete regeneration.

However, urodele amphibians (newts and salamanders) commonly regeneratelimbs and often other organs such as tails, jaws, and parts of the eyethroughout their lifetimes. Such regenerative phenomena are very rareamong reptiles, birds, and mammals as adults, suggesting that the lossof regenerative capacity may have been an adaptive part of theevolutionary transition toward the more advanced tetrapods. severalinvestigators suggested that cells of differentiating muscle and othertissues in the anuran limb lose their ability to revert to theproliferative state and contribute to limb regrowth. Consistent withthis view, regeneration and morphogenesis were found to be enhanced inlimbs of adult frogs when tissue dissociation and cellulardedifferentiation were increased in stump tissues by additional trauma(Polezhaev, 1972). The plasticity of the differentiated state inregeneration-competent limbs and the potential of multinucleate musclefibers to dedifferentiate and re-enter the cell cycle are currentlyactive areas of investigation within the field of limb regeneration(Brockes et al., 2001; Brockes and Kumar, 2002).

Regeneration requires epithelial-mesenchymal interactions at the distallimb stump like those that drive embryonic limb development, and thechanging nature of wound closure after amputation of anuran limbs duringthe transition from larvae to adults has also been studied. Closure oflimb stumps in mammals involves contraction of full-thickness skin andin adult frogs involves rapid formation of connective tissue beneath theapical wound epidermis that initially covers the cut surface (Carlson,1974). Tassava and Olsen (1982) suggested that the inability of highervertebrates to form a functional wound epithelium explains the lostpotential for regeneration. Interfering with distal scar formation inamputated limbs of mammals or adult frogs in order to elicitregeneration have at best been only marginally successful (see review byStocum, 1996), but the importance of establishing proper conditions forthe reciprocal interactions between the apical epithelium and theunderlying mesodermal cells is clear if a limb is to regenerate. Inembryonic limbs, signaling occurs between the apical ectoderm and theadjacent mesodermal cells fibroblast growth factors (FGFs) and theirreceptors. Galis et al. (2003) have suggested that reason for thefailure of limbs of higher vertebrates to re-establish functional tissueinteractions is because limb regeneration is only possible when the limbdevelops as a semiautonomous module not dependent on interactions withtransient structures such as somites.

Reptiles, birds, and mammals limb development begins in the early embryoand involves signaling interactions with various temporary neighboringstructures, while in amphibians limb development occurs much later indevelopment and is not coupled to interactions with transientstructures. Factors and cells from the immune system may also affectregenerative ability (Harty et al., 2003). Development of adaptiveimmunity, which supplements more general and primitive innate immunemechanisms and allows an organism to acquire highly specific defensemechanisms against invading microorganisms, may have yielded immunecells and cytokines whose activity in traumatized tissue is inimical tocell dedifferentiation or the signaling required to initiate limbregeneration, so that the response to injury in the presence of suchimmunity is dominated by tissue repair and fibrosis rather thanregeneration (Mescher and Neff, 2005).

The origin of adaptive immunity during evolution led to the restrictionof regenerative ability is consistent with our knowledge of immunephylogeny (Flajnik et al., 2003). Invertebrates, which usually havewell-developed capacities for regeneration, completely lack adaptiveimmunity. They rely instead on an array of defenses that constitute anextremely effective innate immune system. Mechanisms underlying acquiredor adaptive immunity first appear in jawed vertebrates, becoming moreefficient in various orders of fish and amphibians and highly developedin the homeotherms (Flajnik et al., 2003).

The present invention contemplates to increase the plasticity and alterthe growth potential of cells and tissues by increasing cellulardedifferentiation and tissue dissociation, thereby allowing de novogeneration of cells, tissues and organs. Alterations of immune-responsesby active substances in the extracts are also contemplated.

EXAMPLES Example 1 Cells and Cell Extracts

NCCIT, Jurkat (clone E6-1) and 293T cells (American Type CultureCollection, Bethesda, Md.) are cultured in RPMI 1640 (Sigma, St. Louis,Mo.) with 10% fetal calf serum (FCS), 2 mM L-glutamine, 1 mM sodiumpyruvate and non-essential amino acids (complete RPMI). NIH3T3Swiss-Albino fibroblasts (American Type Culture Collection) are culturedin Dulbecco's modified Eagle's medium (DMEM; Sigma) with 10% FCS,L-glutamine and 0.1 mM β-mercaptoethanol. Mouse ESCs are isolated frominner cell masses of strain sv129 blastocysts and plated on mousefibroblast γ-irradiated feeder layers in ESC medium (DMEM, 15% FCS, 0.1mM β-mercaptoethanol, non-essential amino acids, 1%penicillin/streptomycin) supplemented with 1,000 units/ml (10 ng/ml) ofrecombinant leukemia inhibitory factor (LIF; Sigma) on gelatin-coatedplates. Prior to harvesting for preparing extracts, ESCs are passagedand cultured under feeder-free conditions in RPMI containing 10 ng/mlLIF.

To prepare NCCIT extracts, cells are washed in phosphate buffered saline(PBS) and in cell lysis buffer (100 mM HEPES, pH 8.2, 50 mM NaCl, 5 mMMgCl₂, 1 mM dithiothreitol and protease inhibitors), sedimented at 400g, resuspended in 1 volume of cold cell lysis buffer and incubated for30-45 min on ice. Cells are sonicated on ice in 200-μl aliquots using aLabsonic-M pulse sonicator fitted with a 3-mm diameter probe (B. BraunBiotech, Melsungen, Germany) until all cells and nuclei are lysed, asjudged by microscopy. The lysate is sedimented at 15,000 g for 15 min at4° C. to pellet the coarse material. The supernatant is aliquoted,frozen in liquid nitrogen and stored. Lysate of 95,583±10,966 NCCITcells is used to generate extract. ESC extracts (25-30 mg/ml protein)are similarly prepared from LIF-adapted ESC cultures. 293T, Jurkat andNIH3T3 extracts are also prepared as above. If necessary, extracts arediluted with H₂O prior to use to adjust osmolarity to ˜300 mOsm.

Example 2 Bulge Hair-follicle Stem Cells

To isolate the vibrissa follicles, the upper lip containing the vibrissapad of a subject is cut and its inner surface was exposed. In humanindividuals, hairs from the scalp or other haired body parts may be usedinstead of vibrissa. The vibrissa or hair follicles are dissected undera binocular microscope. The vibrissa are plucked from the pad by pullingthem gently by the neck with fine forceps. The isolated vibrissae werewashed in DMEM-F12 (GIBCO/BRL), containing B-27 (GIBCO/BRL) and 1%penicillin/streptomycin (GIBCO/BRL). All surgical procedures were doneunder a sterile environment. The vibrissa follicular bulge areacontained nestin expressing cells. The cells were isolated by exposureto fluorescent anti-nestin antibodies under fluorescence microscopy. Theisolated cells were suspended in 1 ml of DMEM-F12 containing B-27 with1% methylcellulose (Sigma-Aldrich), and 20 ng·ml⁻¹ basic FGF (bFGF)(Chemicon). Cells were cultured in 24-well tissue-culture dishes(Corning) at 37° C. in a 5% CO₂/95% air tissue-culture incubator. After4 weeks, the bulge-area cells form colonies.

Example 3 Ex Vivo Therapy

Cells to be reprogrammed ex vivo are washed in cold PBS and in coldCa2+- and Mg2+-free Hank's balanced salt solution (HBSS; Invitrogen,Gaithersburg, Md.). Cells are resuspended in aliquots of 100,000cells/100 μl HBSS, or multiples thereof, placed in 1.5 ml tubes andcentrifuged at 120 g for 5 min at 4° C. in a swing-out rotor. Sedimentedcells are suspended in 97.7 ml cold HBSS, tubes placed in a H2O bath at37° C. for 2 min and 2.3 ml SLO (Sigma; 100 mg/ml stock diluted 1:10 incold HBSS) is added to a final SLO concentration of 230 ng/ml. Samplesare incubated horizontally in a H₂O bath for 50 min at 37° C. withoccasional agitation and set on ice. Samples are diluted with 200 mlcold HBSS and cells are sedimented at 120 g for 5 min at 4° C.Permeabilization is assessed by monitoring uptake of a 70,000 Mr Texasred-conjugated dextran (Molecular Probes, Eugene, Oreg.; 50 μg/ml) in aseparate sample 24 h after resealing and replating the cells.Permeabilization efficiency under these conditions is ˜80%.

Following permeabilization, cells to be reprogrammed ex vivo aresuspended at 1,000 cells/μl in 100 ml extract (or multiples thereof)containing an ATP-regenerating system (1 mM ATP, 10 mM creatinephosphate, 25 mg/ml creatine kinase; Sigma), 100 μM GTP (Sigma) and 1 mMof each nucleotide triphosphate (NTP; Roche Diagnostics, Mannheim,Germany). The tube containing cells is incubated horizontally for 1 h at37° C. in a H₂O bath with occasional agitation. To reseal plasmamembranes, the extract is diluted with complete RPMI containing 2 mMCaCl₂ and antibiotics, and cells are seeded at 100,000 cells per well ofa 48-well plate. After 2 h, floating cells are removed and plated cellsare cultured in complete RPMI. The reprogrammed cells can betransplanted back into patient.

Example 4 Cream Base for Use with Cell Extracts

Water - 78% Proteins - 10%

-   -   e.g., Keratin, Filaggrin, and/or Growth factors in trace amounts        (μM-mM amounts of EGF, IGF, IGFII, Insulin, Substance P,        Defensins, NGF)

Lipids—10%

-   -   Squaline 9%, Aliphaic waxes 12%, Sterol esters 33%, Diol esters        7%, Triglycerides 26%, Free sterols 9%, Other lipids 4%.

Cell extract or egg extract or components of extracts—2%

A cream base made from any combination of lipids and/or proteins and/orwater containing cell extracts.

Example 5 Preparation of Fish Egg Extracts

Fresh, unfertilized salmon (Salmo salar) eggs harvested from females inreproductive phase (late fall) are kept on ice, and the extractpreferably made immediately. It is possible to freeze dry eggs in acryoprotectant (e.g., 1.5 M 1.2-propanediol and 0.2 M sucrose) withoutdisrupting the egg membrane. Freezing should be gradual (−1° C./min) to−80° C. Eggs should be thawed and kept on ice throughout the extractpreparation procedure.

Eggs are washed twice in HBSS or seawater with protease inhibitors (10ug/ml). The washing solution is removed and the eggs are lysed andhomogenized in a pre-chilled Dounce glass-glass homogenizator. Thelysate is transferred to Beckman Ultra Clear polyallomer centrifugetubes (5 ml) while avoiding transfer of egg shells, and centrifugatedfor 15 min at 15,000 g at 4° C. in a Beckman ultracentrifuge usingSW55T1 rotor. Three fractions are thereby obtained; lipid top fraction,cytoplasmic middle fraction, and a bottom fraction containing eggshellsand nucleic debris. The cytoplasmic middle fraction is the collectedextract. This extract is expected to contain most cytosolic organellesincluding mitochondria, lysosomes and peroxisomes, should be clear andviscous, and have an orange tint. Protease inhibitors (10 μg/ml stock)are added and extracts are kept at −80° C.

Further fractionation of the cytoplasmic extract is possible.Centrifugation at 100,000 g at 4° C. for 60 minutes yields 2-3fractions, where the top/middle cytoplasmic fraction contains thecytosol with endoplasmic reticulum, SV and microsomes. The extract pH ismeasured by litmus paper, protein concentration measured by Bradfordassay, and osmolarity measured by osmometer.

Mid-blastula Zebra fish embryos are collected, liquid removed and frozento −20° C. To prepare the extract, embryos are thawed on ice, lysed andhomogenized by Dounce glass-glass homogenizator in a small amount ofeither HBSS or seawater (preferably less than 50% liquid v/v). Thelysate is filtered through a sterile linen cloth and centrifugated at5,000 g at 4° C. for 20 minutes in a SX4250 rotor using a Beckman X-22Rcentrifuge. The cytoplasmic extract (supernatant) is collected andprotease inhibitors (10 ug/ml) are added. The extract may be Milliporefiltered (0.22 μm MilliQ sterile filter). The extracts are kept at −80°C. The extract pH is measured by litmus paper, protein concentrationmeasured by Bradford assay, and osmolarity measured by osmometer.

This general procedure is useful for the preparation of extracts fromsea urchin, shrimp, fish eggs/roe or frog eggs. Briefly, roe collectedfrom gravid female fish soon after they liberated their eggs in aspawning program (hCG hormone injected (1 ml/kg) at 6 to 8 hours beforeegg liberation, usually at dawn (2-4 am), or from gravid frogs. Roe/eggsare freeze dried or frozen at −20° C. or used fresh. Roe is collectedfrom different kinds of fish. For sea-urchin, 0.5 M KCl is injectedaround the mouth to evoke shedding of eggs. The extract is prepared fromeggs/roe by crushing (cell cracker or dounce-homogenization) orcentrifugation at different speeds to separate cytoplasm with allcontent, with/without egg-shells (zona pellucida), with/withoutnucleus/cytosol, with/without organelles, with/without lipids. Furtherfractionation can be conducted to isolate one or more of mRNA, proteins,small peptides, carbohydrates and lipids. Major components of fattyacids in the roe are oleic acid, linoleic acid, and omega-3 fatty acids.

Upon application of the above protocol for salmon egg extracts, thesalmon egg extracts had a surprisingly high protein concentrationvarying from 100-380 mg/ml, pH between 6.4-6.8, and an osmolarity ofapproximately 350 mOsm. The extracts were clear and viscous andnon-filterable (by 0.45 μm MilliQ filter). The protein in the extractprecipitated easily upon addition of water or hydrous solutions with lowbuffering capacity due to the high protein content and low pH. Extractscould be neutralized to pH 7.0 by addition of alkaline (1-3 μl M NaOH/mlextract), whereupon dilution in water and hydrous solutions waspossible. Zebra-fish extracts had a protein concentration varying from23-26 mg/ml, pH between 6.4-6.8, and an osmolarity between 80-150 mOsm.The extracts were clear and non-viscous, filterable and diluted readilyin water at all dilutions.

Example 6 Toxicity Testing of Extracts

Extracts with low pH and that contain certain substances may be toxic tocells. Toxicity of each batch should be tested on each cell type that isto be reprogrammed. Cells are harvested and washed twice in HBSS.Approximately 100,000 cells are pelleted and resuspended in 100 ulextract and incubated in a waterbath at 37° C. for 1 hour. Dilutions ofthe extracts may be tested to assess cell survival in extracts ofvarying protein concentration, pH and osmolarity. Optimally, proteinconcentration should be more than 25 mg/ml, pH should be close to 7.2,and osmolarity close to 280 mOsm. Cells and extract are incubated inwells with normal media (as suited to cell type chosen) for 24 hours,and the morphology of the cells inspected by microscopy. Cells areharvested, stained, and viable cells counted. If more than 50% of cellsare non-viable after culture, the extract is considered toxic.

Upon application of the above protocol, 293T cells were viable for atleast 3 weeks after incubation with extracts of salmon eggs and zebrafish embryo with protein concentrations varying from 24-380 mg/ml, atosmolarities between 140-350 and pH 6.9-7.7. At osmolarity below 140mOsm, the cells died.

Cellular morphology of cells reprogrammed with salmon egg extracts orextracts of zebrafish embryos changed after approximately 3 days. 293Tcells become rounder, and some populations of cells start to grow inblastoma like spheres. These changes are persistent, and can be observeduntil 21 days (experiment terminated), although in certain conditionsthe changes seem to reverse towards normal 293T morphology after 2weeks. Upon culture of normal 293T cells with extract added to normalmedia (RPMI-1640 with 10% FCS and 0.2% extract), similar changes inmorphology can be observed as seen for reprogrammed cells cultured innormal media. Additionally, cells cultured with salmon egg extracts inparticular have an increased growth rate compared to normal cells. Whenstarving cells (RPMI-1640 with 0.5% FCS), growth rate decreasessignificantly for non-extract treated cells, and morphology of cellschanges slightly. For starved cells grown with extracts (0.2% extract instarvation media), the changes are more pronounced. In this case, mostcell populations grow in blastomere like spheres, and the spheres detachfrom the culture vessel and float in the media, where they keep growing.Interestingly, the deceleration in growth rate is reversed in cellscultured with extract added to the starvation medium.

Example 7 Gene Expression Assays of Extracts

To verify extract expression of genes to be studied in reprogrammedcells, RT-PCR may be conducted on RNA isolated from extracts. RNA may beisolated from extracts by the method of choice, for example by using aQiagen RNeasy Plus Kit (Qiagen). The RNA is quantified byspectrophotometer, and stored at −80° C. 1 μg RNA is used for cDNAsynthesis. cDNA synthesis may be conducted for example, by using theiScript cDNA Synthesis Kit (Bio-Rad), followed by PCR conducted withprimers of choice. Positive controls are included for each primer settested. PCR products are run on a 1% agarose gel with ethidium bromideand bands visualized by UV lamp.

Upon application of the above protocol, PCR products of extractsobtained by specific primers for human genes of interest as seen onagarose gels were compared to bands obtained from positive control humancell lines previously shown to express genes herein investigated (e.g.,OCT4, NANOG, SOX2, UFT1, GAPDH, REX1 (a.k.a. ZFP42), LMN-A, LMN-B1,OXT2, ACI33, APL and STELLA). Positive control NCCIT cells showed asingle band at the expected size for each gene tested, while PCRproducts from neither salmon egg extracts nor zebra fish embryo extractsgive bands on the gel. These results indicate that the extracts do notexpress the human gene variants detected with the primers used.

Micro array data of fibroblasts stimulated 8 days with extract showup/down regulation of a number of genes compared to untreated cells.These genes include but are not limited to several genes previouslyshown to be involved in the process which is related to woundhealing/cell regeneration. Examples of genes regulated by the extractare; extra cellular matrix proteins like collagen I and III, fibronectinand elastin; enzymes like matrix metalloproteinases; growth factors likeplatelet derived growth factor (PDGF), transforming growth factor beta(TGF-□□, vascular endothelial growth factor (VEGF), interleukins (ILs);the cell differentiation marker smooth muscle actin; and chemokines likethe chemokine (C-X-C motif) ligand (CXCLs).

Examples of gene regulation in human skin fibroblasts (hSF) afterextract treatment are provided in Table 1.

TABLE 1 COL11A1 49 down collagen, type XI, alpha 1 [Homo sapiens]COL12A1 5.2 up collagen, type XII, alpha 1 [Homo sapiens] COL13A1 6.8 upcollagen, type XIII, alpha 1 [Homo sapiens] COL14A1 17.8 up collagen,type XIV, alpha 1 [Homo sapiens] COL15A1 25.4 up collagen, type XV,alpha 1 [Homo sapiens] COL16A1 45 up collagen, type XVI, alpha 1 [Homosapiens] COL17A1 132.4 down collagen, type XVII, alpha 1 [Homo sapiens]COL18A1 31.8 down collagen, type XVIII, alpha 1 [Homo sapiens] COL19A196.3 down collagen, typeXIX, alpha 1 [Homo sapiens] COL1A1 303.9 upcollagen, type I, alpha 1 [Homo sapiens] COL1A2 20.1 up collagen, typeI, alpha 2 [Homo sapiens] COL20A1 53.1 down collagen, type XX, alpha 1[Homo sapiens] COL21A1 83.5 down collagen, type XXI, alpha 1 [Homosapiens] COL22A1 4.4 down collagen, type XXII, alpha 1 [Homo sapiens]COL23A1 21.3 down collagen, type XXIII, alpha 1 [Homo sapiens] COL25A1 4down collagen, type XXV, alpha 1 [Homo sapiens] COL3A1 207 up collagen,type III, alpha 1 [Homo sapiens] COL4A1 8.9 down collagen, type IV,alpha 1 [Homo sapiens] COL4A4 7.4 down collagen, type IV, alpha 4 [Homosapiens] COL4A5 6.4 down collagen, type IV, alpha 5 [Homo sapiens]COL4A6 91.6 down collagen, type IV, alpha 6 [Homo sapiens] COL5A1 193.7up collagen, type V, alpha 1 [Homo sapiens] COL5A3 13.8 down collagen,type V, alpha 3 [Homo sapiens] COL6A1 17.6 up collagen, type VI, alpha 1[Homo sapiens] COL6A2 6.1 down collagen, type VI, alpha 2 [Homo sapiens]COL6A3 35.9 up collagen, type VI, alpha 3 [Homo sapiens] COL8A1 9.2 upcollagen, type VIII, alpha 1 [Homo sapiens] COL8A2 5.1 down collagen,type VIII, alpha 2 [Homo sapiens] COL9A1 28.2 down collagen, type IX,alpha 1 [Homo sapiens] COL9A2 15.6 down collagen, type IX, alpha 2 [Homosapiens] COL9A3 13.7 down collagen, type IX, alpha 3 [Homo sapiens] COLQ22.7 down collagen-like tail subunit of asymmetric acetylcholinesterase[Homo sapiens] ELN 130.9 down elastin [Homo sapiens] FN1 4.7 upfibronectin 1 [Homo sapiens] HABP2 4.4 down hyaluronan binding protein 2[Homo sapiens] HABP4 8 up hyaluronan binding protein 4 [Homo sapiens]HAS2 4.9 up hyaluronan synthase 2 [Homo sapiens] HYAL1 4.1 downhyaluronoglucosaminidase 1 [Homo sapiens] HYAL3 3.5 uphyaluronoglucosaminidase 3 [Homo sapiens] HYAL4 25.6 downhyaluronoglucosaminidase 4 [Homo sapiens] ACTA2 20.4 up actin, alpha 2,smooth muscle, aorta [Homo sapiens] EGF Not epidermal growth factor(beta-urogastrone) listed [Homo sapiens] TGFB1 19.7 down transforminggrowth factor, beta 1 [Homo sapiens] TGFB1/1 9.1 up transforming growthfactor, beta 1 [Homo sapiens] TGFBI 107.1 up transforming growth factor,beta-induced, 68 kDa [Homo sapiens] TGFBR2 20.9 up transforming growthfactor, beta receptor II (70/80 kDa) [Homo sapiens] TGFBR3 50.5 uptransforming growth factor, beta receptor III [Homo sapiens] TGIF1 20.8up TGFB-induced factor homeobox 1 [Homo sapiens] VEGFA 95.9 downvascular endothelial growth factor A [Homo sapiens] VEGFB 82 up vascularendothelial growth factor B [Homo sapiens] VEGFC 11.6 up vascularendothelial growth factor C [Homo sapiens] PDGFD 19.3 up plateletderived growth factor D [Homo sapiens] PDGFRA 64.1 up platelet-derivedgrowth factor receptor, alpha polypeptide [Homo sapiens] PDGFRB 66.9 upplatelet-derived growth factor receptor, beta polypeptide [Homo sapiens]IL1B 20.7 down interleukin 1, beta [Homo sapiens] IL1R1 3.5 upinterleukin 1 receptor, type I [Homo sapiens] MMP14 3.6 down matrixmetallopeptidase 14 (membrane-inserted) [Homo sapiens] MMP16 15 downmatrix metallopeptidase 16 (membrane-inserted) [Homo sapiens] MMP17 6.5down matrix metallopeptidase 17 (membrane-inserted) [Homo sapiens] MMP193.6 down matrix metallopeptidase 19 [Homo sapiens] MMP2 4.1 up matrixmetallopeptidase 2 (gelatinase A, 72 kDa, gelatinase, 72 kDa type IVcollagenase) [Homo sapiens] MMP20 8.4 down matrix metallopeptidase 20[Homo sapiens] MMP23A 9 down matrix metallopeptidase 23A (pseudogene)[Homo sapiens] MMP25 11.4 down matrix metallopeptidase 25 [Homo sapiens]MMP28 3.1 down matrix metallopeptidase 28 [Homo sapiens] TNF Not tumornecrosis factor (TNF superfamily, listed member 2) [Homo sapiens] IFNGNot interferon, gamma [Homo sapiens] listed IL12A Not interleukin 12A(natural killer cell stimulatory listed factor 1, cytotoxic lymphocytematuration factor 1, p35) [Homo sapiens] IL12B 55.3 down interleukin 12B(natural killer cell stimulatory factor 2, cytotoxic lymphocytematuration factor 2, p40) [Homo sapiens] IL18 39.6 up interleukin 18(interferon-gamma-inducing factor) [Homo sapiens] IL23A Not interleukin23, alpha subunit p19 listed [Homo sapiens] CXCL1 Not chemokine (C-X-Cmotif) ligand 1 (melanoma listed growth stimulating activity, alpha)[Homo sapiens] CXCL10 Not chemokine (C-X-C motif) ligand 10 listed [Homosapiens]

Example 8 Reprogramming of Cells with Fish Egg or Embryo Extracts

The cell type of choice (e.g., human 293T cells and adipose stem cells(ASC tested)) is harvested, kept on ice, and washed twice in ice coldHBSS. Approximately 100,000 to 500,000 cells are pelleted bycentrifugation (300 g, 4° C. for 10 minutes). Cells may be permeabilizedwith streptolysin-O (SLO) by incubation for 50 minutes in a 37° C.waterbath prior to reprogramming, however this is not necessary forreprogramming effects of fish egg or embryo extracts. After SLOincubation, cells are washed in ice cold HBSS, centrifugated and excessliquid removed from pellet. Cells are resuspended in 100 ul extract per100,000 cells and incubated for 1 hour at 37° C. in a waterbath.Approximately 100,000 cells are seeded in wells with complete media ofchoice. If SLO permeablization has been conducted, cells are cultured inmedia with 2 mM CaCl₂ for 2 hours after reprogramming to reseal the cellmembranes. The media should be changed 2-12 hours after reprogramming.To assess extent of permeabilization by SLO, use epifluorescentmicroscopy for cells incubated for 50 min in 0 or 100 ng/ml SLO with 50ug/ml Alexia red-conjugated dextran (10,000 M_(r) or 70,000 M_(r)dextran) to verify cell permeabilization and resealing.

Cells are cultured in wells until proliferation allows splitting tolarger vessels. Split cells as appropriate for the cell type, but do notallow them to become confluent. Pellets for gene-analysis should becollected weekly and morphology assessed by phase-contrast microscopy ateach passage. Cells can be cultured as long as desired, but to assesslasting reprogramming effects, 40 days is a suggested minimum.

Additionally, cells may be reprogrammed by incubation in media enhancedwith fish egg or embryo extracts. Cells are reprogrammed by adding 0.4%extract to normal complete medium (10% FCS) or starvation media (0.5%FCS). Cells of choice are grown to 50% confluency, and normal mediumreplaced with complete medium or starvation medium containing 0.4%extract. Split cells as appropriate with media containing extract. Freshmedium with extracts should be added to cells at least two times perweek if splitting less than twice weekly. Pellets for gene-analysisshould be collected weekly and morphology assessed by phase-contrastmicroscopy at each passage. Cells can be cultured as long as desired,but to assess lasting reprogramming effects, 40 days is a suggestedminimum.

Upon following the protocols listed above, cells reprogrammed withfish-egg extracts or zebra-fish embryo extracts, or grown in media withextracts added, were harvested and RNA isolated. Reprogrammed or normal293T cells were incubated in either complete media (RPMI-1640 with 10%FCS) with/without extracts (0.2%), or in starvation media (RPMI-1640with 0.5% FCS) with/without extracts. Real-Time RT-PCR was run to studyup- and down-regulation of differentiation marker genes. After 7 days, apronounced up-regulation in the OCT 4 gene is seen in extract treatedcells, and the changes are still seen after 17 days. Gene expression wascalculated with the housekeeping gene GAPDH as reference of geneexpression and can be seen in Tables 2 and 3. Values represent increasein gene expression in treated cells over untreated cells grown in normalmedia. Values given are for cells treated for 17 days, 17 days afterreprogramming.

TABLE 2 Reprogrammed Reprogrammed w/0.2% w/salmon egg w/zebra fishNormal w/0.2% zebra fish Reprogrammed Reprogrammed extract, grown embryoextract, media salmon egg embryo w/salmon egg w/zebra fish with 0.2%grown with 10% FCS extract extract extract embryo extract extract 0.2%extract OCT4 59.78 100.58 34.98 18.20 82.26 90.12 NANOG 3.83 4.66 1.300.70 3.82 1.58 SOX2 5.58 6.16 4.24 8.36 3.67 2.41

TABLE 3 Reprogrammed w/0.2% w/0.2% Reprogrammed w/zebrafish Starvation293T salmon zebra fish Reprogrammed Reprogrammed w/salmon egg embryoextract, media cells, egg embryo w/salmon egg w/zebra fish extract,grown grown with 0.2% 0.5% FCS untreated extract extract extract embryoextract with 0.2% extract extract OCT4 0.20 11.69 6.45 5.17 12.95 9.322.78 NANOG 0.14 0.51 0.41 0.37 1.10 0.32 0.14 SOX2 0.63 4.34 1.67 2.662.47 4.49 1.12Results show an up-regulation (18 to 100 times) of the OCT-4 gene in allcells treated with extracts compared to untreated cells. The changes inNANOG gene expression are more modest, with an up-regulation varyingfrom none to 5 times. For SOX2 gene expression, the up-regulation seenvaried from 2 to 8 times over basal.

In starved cells cultured without extracts, all OCT4, NANOG and SOX2genes are down-regulated (0.2-0.6 times of normal 293T cells grown innormal medium). Adding 0.2% extract to the starvation media rescues thegene expression profile, and up-regulates OCT4 gene expression from 5 to13 times over normal 293T cell expression—and up-regulation of approx.100 times from untreated, starved 293T cells. The same is not seen forNANOG gene expression, where the down regulating effect of starvation isnot rescued. For SOX2 gene expression, a rescue similar to that of OCT4is observed, although not as marked (up to 4 times up-regulated). Inthese experiments, salmon egg extracts seem to give the largest rescueand up-regulation of the dedifferentiation-associated genes.

Replications of these reprogramming experiments in triplicate yieldedresults confirming that salmon egg extracts upregulate dedifferentiationassociated genes, indicating increased “stemness” of the 293T cells.

Reprogramming was conducted by three different methods:

1. Reprogramming as described in methods, followed by normal cellculture;

2. Reprogramming as described followed by culture in media supplementedwith 0.4% salmon egg extract (same as used for reprogramming); and

3. Normal cells not reprogrammed, cultured in media supplemented with0.4% salmon egg extract (same as used for reprogramming).

All three methods yield changes in morphology and gene expression incells, but at different levels and occurring at different times. Geneexpression changes are seen at the same time as morphological changesare observed, varying from day 5 after reprogramming to day 28 afterreprogramming. This seems to be dependent on the method used:reprogramming (methods 1 and 2) may yield more rapid changes thannot-reprogramming and culturing in supplemented media (method 3).

Reprogrammed cells with morphological changes and gene expressionchanges as shown below, were additionally labeled with OCT4 and NANOGantibodies and visualized with fluorescent secondary antibodies in aconfocal microscope to verify increased expression of these genes.

Example results shown in Tables 4 and 5 below, where numbers representfold up- or downregulation of the dedifferentiation associated genesOCT4, NANOG and SOX2 as compared to normal 293T cells. Numbers below oneindicate downregulation, and numbers over one indicate up regulation.Large up regulation effects are marked in blue in the table. The upregulation of SOX2 occurs at day 5 in adipose stem cells, only minorchanges in gene expression can be detected in the controls. This is inagreement with experiments conducted with different extracts(unpublished observations, Taranger et al., 2006).

TABLE 4 ASC REPROGRAMMING DAY OCT4 NANOG SOX2 Reprogramming A1, normalmedia 5 0.257 0.071 NA 11 0.74 0.47 3.2  19 0.78 1.17 NA 40 1.27 0.22 NAReprogramming A2, media with 5 0.88 0.17 2.81 0.4% extract ReprogrammingB1, normal media 5 0.78 0.79 NA 11 0.37 0.07 1.11 18 2.25 1.91 NA 351.27 0.12 NA

TABLE 5 DAY OCT4 NANOG SOX2 293T CELL REPROGRAMMINGS Reprogramming A1,normal media 11 24.99 382.08 2.73 25 2.77 19.54 7.01 34 20.5 228.13 1.4442 1.31 0.82 3.27 293T Reprogramming A2, media with 11 0.92 5.92 0.930.4% extract 25 0.9 0.76 0.99 34 1.34 123.61 1.84 42 8.14 2.69 6.55Reprogramming B1, normal media 27 2.22 2.74 1.69 Reprogramming B2, mediawith 27 2.51 3.28 1.80 0.4% extract Reprogramming C1, normal media 50.41 0.53 1.84 11 0.61 0.56 1.73 20 8.65 14.83 1.89 28 125.70 18.0384.69 Reprogramming C2, media with 5 0.92 0.60 1.61 0.4% extract 11 0.651.10 1.17 20 10.12 45.30 1.84 28 2.25 0.79 5.61

The studies presented herein, give proper protocols for preparing fishegg extracts, characterization and toxicity tests of such extracts,protocols for cell reprogramming with the extracts, as well as resultsof changes induced by the extracts upon cells. The results includemorphological changes presented as microscopic images, as well aschanges in gene expression in the treated cells presented as real-timePCR data. Reprogramming of 293T cells has been conducted 17 individualtimes, with changes in morphology observed in 12 of the 17reprogrammings. Alterations in gene expression has been observed in 8 of12 studied. Alterations in morphology are correlated with changes ingene expression, i.e., changes in morphology occur at the same time asgene expression changes in the cells, and this is verified byimmunofluorescent labeling of dedifferentiating genes in thereprogrammed cells. Reprogramming of adipose stem cells has beenconducted 6 times, and morphological changes has only been observed in 1of these. Only minor changes in gene expression changes in thesereprogrammed cells can be detected.

Example 9 Morphological Changes in Cells Treated with Extracts

Morphology of cells reprogrammed with salmon egg extracts or extracts ofzebra-fish embryos change after approx. 3 days. 293T cells becomerounder, and some populations of cells start to grow in blastoma likespheres. These changes are persistent, and can be observed until 21 days(experiment terminated), although in certain conditions the changes seemto reverse towards normal 293T morphology after 2 weeks. Upon culture ofnormal 293T cells with extract added to normal media (RPMI-1640 with 10%FCS and 0.2% extract), similar changes in morphology can be observed asseen for reprogrammed cells cultured in normal media. Additionally,cells cultured with salmon egg extracts in particular have an increasedgrowth rate compared to normal cells. When starving cells (RPMI-1640with 0.5% FCS), growth rate decreases significantly (not shown) fornon-extract treated cells, and morphology of cells changes slightly. Forstarved cells grown with extracts (0.2% extract in starvation media),the changes are more pronounced. Here, most cell populations grow inblastomer like spheres, and the spheres detach from the culture vesseland float in the media, where they keep growing.

Interestingly, the deceleration in growth rate is reversed in cellscultured with extract added to the starvation medium. Successfulreprogrammings commonly grow in large clumps (>2 mm diameter) visible inthe cell vessels with the naked eye.

Example 10 Alterations in Growth Patterns in Cells Incubated withExtracts

500,000 293T cells were seeded in medium sized round culture dishes, andincubated in normal media or media with addition of extract orstarvation media. Cells were harvested after 24, 41, and 68 hours,counted and the growth rates were calculated. The results are presentedTables 6 and 7.

TABLE 6 Calculate nr of cell divisions: Number of cells (Ne) = Initial(No)/2{circumflex over ( )}number of divisions (g) number of cells 24 h41 h 68 h min/generation = 60314-24 060313-0 (T(hours) × 60)/g g = 24 Hlog Ne log No Min/gen = 2.3704 517000 100000 293norm 607.4923 768.2811693.4967 2.018123 405000 100000 293Tnorm + LE 713.5343 824.4037 922.37861.613692 306000 100000 293Tnorm + ZE 892.3634 824.9083 689.2529 2.208113462000 100000 293Tstarv 652.1406 1072.826 1695.597 2.,170141 450000100000 293Tstarv + LE 663.5513 989.671 669.0016 2.3704 517000 100000293Tstarv + ZE 607.4923 1252.756 1228.08 60315-41 060313-0 g = 41 H logNe log No 3.201953 920000 100000 293norm 2.983975 791000 100000293Tnorm + LE 2.98215 790000 100000 293Tnorm + ZE 2.29301 490000 100000293Tstarv 2.485675 560000 100000 293Tstarv + LE 1.96367 390000 100000293Tstarv + ZE 60314-68 060313-0 g = 68 H log Ne log No 5.883229 5900000100000 293norm 4.423347 2145000 100000 293Tnorm + LE 5.919453 6050000100000 293Tnorm + ZE 2.406232  530000 100000 293Tstarv 6.09864 6850000100000 293Tstarv + LE 3.322259 1000000 100000 293Tstarv + ZE Generationtime: T: Time elapsed between No (near beginning of exponential growth)and (near end of exponential Ne growth) Growth curve: Log#cells g = (logNe − log No)/0.301

TABLE 7 Condition 0 H 24 H 41 H 68 H 293norm 5 2.783541 2.88552 2.841044293Tn + LE 5 2.853415 2.91614 2.964909 293Tn + ZE 5 2.950542 2.9164062.838379 293Tstarv 5 2.814341 3.030529 3.229323 293Ts + LE 5 2.8218742.995491 2.825427 293Ts + ZE 5 2.783541 3.097867 3.089227

Growth rate changes are seen, where starved cells grow much slower thancells in normal media throughout the study. This effect is rescued byaddition of salmon egg media after 48 hours of culture. The cells grownin normal media with zebra-fish embryo extract and salmon-egg extractgrow fastest of all the cells during the first 24 hours. See FIGS. 1-3.

Example 11 Enhanced Wound Healing with Salmon Egg Extracts

Aim of study: Investigate the effects of salmon egg extract developed onwound healing in the skin of mice.

Methods:

Summary: Two types of wounds were induced in the dorsal skin of mice. Anexcision wound of 1 cm diameter was induced on the left side of the backof each mouse (n=12 repeated 3×), and an incision wound of 2 cm lengthwas induced on the right side of the back parallel to the spine. Halfthe mice (picked randomly) were treated with 30 μl salmon egg extractafter wound induction and every 3 days for 12 days. The control groupreceived no treatment. Wounds on all mice (treated and controls) weresprayed with liquid Band-Aid on day 1 in one of the 3 repetitions, butthis did not affect the differences seen between the control and treatedmice as observed in all 3 experiments (with/without spray bandage).

In each experiment, mice were divided in 3 cages, each with 2 controlsand 2 treated animals. The wound healing process was monitored over 12days, with measures taken including wound areas, days until completehealing (reepithelialization and loss of scab), and size of resultingscar. Biopsies were taken on day 1 and day 12 for further analysis, andwounds were photographed regularly to document healing progress.

Materials:

Mice: A/J or NMRI albino males.

Ethanol for sterilization of skin prior to wound induction.

Small surgical scissors and micro scissors, surgical blades andtweezers.

Salmon egg-extract, batch LE4, prepared as described above.

Isofluran gas: FORENE Isofluran Vnr 506949, lot 22397VA, exp 2009-10(Abbott, Solna, Sweden)

Vaporizer: Datex-Ohmeda Isotec 5

Liquid nitrogen for biopsy samples.

4% PFA (in PBS) for biopsy samples.

Digital camera to take pictures of wounds and skin during healing.

1 cm diameter round mold for inking on wound size in animals.

Study Design:

Animals. Healthy inbred male NMRI or A/J mice (separate studies),weighing between 25 g and 35 g were obtained from the animal house ofthe Institute of National Public Health, Oslo, Norway. The mice wereacclimatized for one week prior to the experiments, and housed inpolypropylene cages on normal food and water ad libitum, and wereear-labeled (1-4 in each cage) one week prior to start of theexperiments. Animals were periodically weighed before and afterexperiments. The mice were anaesthetized prior to infliction of theexperimental wounds. The surgical interventions were carried out understerile conditions using isofluran gas (oxygen+isofluran mixed invaporizer). Animals were closely observed for any infection; those whichshowed signs of infection were separated and excluded from the study. Anacute toxicity study was conducted for the extracts as describedelsewhere in this patent. The study was approved by the Ethics Committeeof Norway.

Wound healing activity. Excision and incision wound models were used toevaluate the wound-healing activity of salmon egg extracts. Each animalreceived an excision wound on the right side of the back, and anincision wound on the left side on the back. The wounds were induced onday 1 and the study terminated on day 12.

Excision and incision wounds. Each mouse was inflicted with one excisionwound (Morton J J P, Malone M H. Evaluation of vulnerary activity by anopen wound procedure in rats. Arch Int Pharmacodyn. 1972; 196:117-126)and one incision wound (Ehrlich H P, Hunt T K. Effect of cortisone andvitamin A on wound healing. Ann Surg. 1968; 167:324-328.). The mice wereanaesthetized prior to creation of the wounds, with isofluran gas (bymask, system details below). The dorsal fur of the animal was shavedwith electric clipper and the area of the excision wound to be createdwas outlined on the left side of the back of the animals withwaterproof, permanent marker. An excision wound of 1 cm in width(circular area=0.785 cm2) of full skin thickness (app. 1 mm) was createdalong the markings using toothed forceps, a surgical blade and pointedscissors, the entire wound left open. On the right side of the spine, alongitudinal paravertebral incision of 2 cm long was made through theskin and cutaneous tissue on the back. The groups of experimentalanimals were treated with aqueous salmon egg extracts (30 μl), topicallyapplied to the wound every third day. The control group wounds were leftuntreated.

The parameters studied were wound closure, wound size, scar size,epithelialisation time, and histology (morphological parameters of theskin). The measurements of the wound areas of the excision wound modelwere taken on 1st, 5th, 9th and 12th day following the initial woundusing transparent paper and a permanent marker. The wounds on each mousewere photographed daily with a digital camera (see details below). Theperiod of epithelialization was calculated as the number of daysrequired for the wounds to become scab free.

Biopsies. On day 1, the skin excised in the creation of the excisionwound was kept as a normal skin biopsy, allowing each animal to be itsown control in later biopsy analysis. One half of the biopsy was fixedin 4% PFA, the other snap-frozen in liquid nitrogen. In the excision andincision wound model, granulation tissue formed on the wound was excisedon the 12th postoperative day, after the termination of the animals bycervical dislocation or CO₂ gas. Excision and incision wounds weresurgically removed along the initial wound induction markings. One halfof the biopsy was fixed in 4% PFA, the other half snap-frozen in liquidnitrogen for later analysis.

Histopathological analysis of biopsies. Half of the healing tissuesobtained on the 12th day from all animals in the excision and incisionwound model was fixed in paraformaldehyde (4% in sodium-phosphatebuffer) for 2 hours at room temperature and stored at 4 degreesCentigrade, sectioned on a cryostat antiparallel to the skin surface.Sections were stained with H&E and parameters of the scar, including thethickness of the granulation tissue, were measured in the microscope.

Standard procedure for H&E staining was conducted. Briefly, 10 umcryostat sections of mouse skin biopsies (on SuperFrost Plus slides)were rehydrated (from absolute through 96% and 70% ethanol) beforecoloring with hematoxylin (7 min) (Sigma 51275 HEMATOXYLIN SOLUTION ACC.TO MAYER), washed in running water (5 min), colored with eosin (1 min)(Sigma HT110116 EOSIN Y SOLUTION ALCOHOLIC), rinsed shortly in water anddehydrated (from 70% through 96% to absolute ethanol followed by 2×5 minin xylol). Sections were mounted directly from xylol with Eukitt (Sigma03989-100ML EUKITT® QUICK-HARDENING MOUNTING ME-DI).

H&E stained sections of mouse skin biopsies taken at day 1 (at time ofwound induction) and day 12 (post healing) from representative treatedand control animals were studied in the light microscope, digitalpictures taken with the 4×, 10×, 20× and 40× objectives and measures ofskin thickness and scar parameters were taken from the digital images.

Immunolabeling of cryostat sections of mouse skin biopsies. Sections ofexcision wound biopsies taken at day 1 and day 12 of representativeextract treated and untreated controls were doubly immunolabeled aspreviously described (Boulland et al. Expression of the vesicularglutamate transporters during development indicates the widespreadcorelease of multiple neurotransmitters. J Comp Neurol. 2004 Dec. 13;480(3):264-80) with antibodies against either NANOG (rabbit polyclonal,Abcam) and calbindin (mouse, Abcam) or OCT3/4 (rabbit polyclonal, SantaCruz) and calretinin (goat, Chemicon) to look for increased expressionof stem cells (as indicated by presence of NANOG and OCT3/4) in thehealed wounds.

Results

Healing rates of wound treated with salmon egg extract compared tountreated controls.

Visual appearance of treated and untreated skin wounds. Results indicatea speedier wound healing in the extract treated animals (images notshown), with significant differences at day 9 and 12. Furthermore, woundsizes reduced more rapidly reduced in the extract treated animals, withsignificant differences at day 5 and 9. See FIG. 4.

Hematoxylin-Eosin staining of skin biopsies for histological examination(paraffin embedded or cryostat sections). Biopsies taken from the skinremoved to form the excision wound at day one and comparable biopsies ofthe same area taken at day 12 from 6 animals were cryostat sectioned,stained with H&E and microscopy images were taken and analyzed.Morphologically, sections of biopsies of normal skin from day 1 of thecontrol and treated animal groups were similar, with equal measures ofskin parameters. At day 12, the scar tissue, particularly the collagenorganization, appeared more disorganized in the control animals comparedto the extract treated animals.

TABLE 8 Distance Total skin Epidermal Dermal Wound/scar betweenthickness thickness thickness diameter hair sacs (um) (um) (um) (um)(um) Controls Day 1 391 15 340 10000 254 Day 12 1154 63 838 1913 144Treated Day 1 389 17 326 10000 218 Day 12 989 46 780 2113 157

Measurements (average of 3 independent measurements per section) weretaken as follows: Epidermal thickness was measured from the stratumgerminatum (basal keratinocytes) to the stratum corneum. Dermalthickness was measured from below the stratum germinatum to the subcutis(adipose tissue below dermis). Total skin thickness was measured ascombined thickness of epidermis and dermis. The scar diameter at day 12was measured between the wound healing tongues on either side of thescar tissue, and compared to the day 1 excision wound diameter (1 cm).Distance between hair sacks was measured between the centers of the hairpapillae of adjacent hairs (proximal to the scar at day 12).

The measurements show that there is less variance in scar thickness,diameter, epidermal and dermal thickness in treated animals compared tothe untreated controls. The extract treated animals displayed a morenormal epidermal thickness (closer to epidermal thickness as measured inthe same animals at day 1), and the distance between the newly formedhair sacs proximal to the scar were more evenly distributed (moresimilar to hair sack distribution in normal skin at day 1), while thehair sacs of the healed skin in control animals was more disorganizedand distributed with a smaller distance between hair sacs compared tonormal skin.

In summary, the data show that extract treated animals have 41% thinnerscar (total skin thickness at centre of scar) compared to untreatedcontrols, and the newly formed epithelium in the healed skin is 148%thinner, and the new dermis 7% thinner in treated animals compared tountreated controls. Additionally, the diameter between new hair sacs is16% closer to pre-operative distance in treated animals compared tountreated controls.

Immunolabeling of cryostat sections of mouse skin biopsies. Sections ofexcision wound biopsies taken at day 1 and day 12 of representativeextract treated and untreated controls were doubly immunolabeled aspreviously described (Boulland et al.) with antibodies against eitherNANOG (rabbit polyclonal, Abcam) and calbindin (mouse, Abcam) or OCT3/4(rabbit polyclonal, Santa Cruz) and calretinin (goat, Chemicon) to lookfor increased expression of stem cells (as indicated by presence ofNANOG and OCT3/4) in the healed wounds.

OCT3/4 is a marker of embryonic and other stem cells, foundpredominately in the nucleus. OCT4 (green) staining was detected in thebasal layer of epidermis (proliferating keratinocytes) (novel finding).Interfollicular keratinocytes in culture have previously beentransfected with OCT-4 which resulted in increased expression of Sox-2,Nanog, Uft1 and Rex-1.

NANOG expression often follows the expression of OCT4 in stem cells.NANOG labeling was detected at the base of hair sacks (hair stem cells)as well as in migrating cells along the wound healing tongue at day 12(novel found)

CALRETININ is a calcium-binding protein shown to be present in thecompanion cell layer of the human hair follicle. Calretinin staining wasseen along the hair shafts as expected.

CALBINDIN is found in the nucleus and cytoplasm of epidermalkeratinocytes (higher in nucleus than in cytoplasm). Upon wounding, thelevels of calbindin in the nucleus drop for approx. 10 days postwounding. Calbindin labeling was seen in keratinocytes (epidermis andlayer around hair shafts).

Further analysis using Z-stacks and Fourier transformation with confocalmicroscope will be necessary to look for differences between the treatedwounds and the untreated controls.

Example 12 Reprogramming of Human Skin Fibroblasts and HEK Cells

Subculture of hsF Cells (Human Skin Fibroblasts).

Complete media for hsF

-   -   500 ml DMEM F-12 (+Glutamax)    -   50 ml (10%) FCS (Fetal Calf Serum—heat inactivated)    -   5 ml (1%) PenStrep

Starvation Media for hsF

-   -   500 ml DMEM F-12 (+Glutamax)    -   5 ml (1%) PenStrep

Culture the cells in large flasks (162 cm2)—ca 1 mill cells per largeflasks at confluence, or on coverslips in wells for reprogramming. (hsFcells used were from ACCT).

Subculture hsF Cells:

1. Rinse the cell layer twice with 10-15 ml PBS to remove all traces ofserum.

2. Add 2 ml Trypsin-EDTA solution until cell layer is dispersed (5-7minutes).

3. Add min 4 ml media and aspirate cells by gently pipetting.

4. Subculture ratio 1:2-1:4; Add 2-3 ml of the cell suspension to theflask and fresh media to total 25 ml. Subculture the cells 1:2 to 1:4every 2 to 3 days—e.g. 1:4 twice a week.

Freeze Cell-pellet:

1. Make fresh freeze-media:

-   -   a. Normal media with 20% FCS and 10% DMSO

2. Follow the protocol above (subculture) to point 3; thereafter

3. Transfer cells to a 50 ml Nunc-tube and spin at 300 g (1500 rpm), 10min 4 C.

4. Resuspend cells to 1 million per 1 ml freeze media and aliquot 1 mlto Nunc cryo tubes.

5. Freeze the cells in Mr. Frosty-box with isopropanol at −80° C. overnight (−1° C./min).

6. Transfer to nitrogen tank.

Make Pellets for RNA-isolation:

Follow the subculture protocol to point 3; thereafter

1. Transfer cells to a 50 ml Nunc-tube and spin at 300 g (1500 rpm), 10min 4 C.

2. Wash cells in one ml ice cold PBS per million cells and spin at (300g, 10 min 4° C.).

3. Resuspend the pellet in the same amount PBS and add 1 ml to eppendorftube for RNA pellets.

4. Spin at 300×g 10′ at 4° C.

5. Aspirate PBS.

6. Keep pellet on ice and snap-freeze in liquid N2.

7. Transfer to −80° C.-freezer.

Reprogramming hsF on Coverslips, Including SLO

Objective: To reprogram cells with a nuclear-free extract to alter geneexpression, morphology and elements of growth and to study changes instate of differentiation.

Materials: hsF cells grown in 24-well plate on coverslips (ca 100.000cells sown out per well in starvation media ca 5 days earlier and ca50.000 cells sown out per well in normal media ca 3 days earlier);Extract (salmon egg extract); Incubation in media for control; 1×PBS;Ca²+ free Hanks Balanced Salt Solution (HBSS) at 4° C.; TE to loosen hsFcells from flask; SLO stock of 100 μg/ml diluted 1:100 in HBSS; ATP (200mM stock in water); GTP (10 mM stock in water); Phosphocreatine (2Mstock in water); Creatine kinase (5 mg/ml stock in water); Autoclaved MQwater; Waterbath at 37° C.; CaCl₂ (2 mM) enriched medium: 100 mM CaCl2stock is prepared by mixing 1.67 g CaCl2 with 15 ml distilled water andsterile filtered. 2 mM concentration of CaCl2 is made by e.g. mixing 50μl of 100 mM CaCl2 with 2450 μ1 of reprogramming medium.

Procedure: Wash cells twice in ice cold 1×PBS (1 ml). Wash cells twicein cold HBSS (1 ml). Preheat samples in incubator, 37° C., 2-3 min andremove HBSS. Add 110 μl HBSS and 90 μl SLO (to a final SLO concentrationof 450 ng/ml) and mix. Add 200 μl HBSS to control wells without SLO.Incubate in incubator 30 min, tilt the plate every 10 min. Remove theSLO (keep one parallel where SLO remains in the well). Prepare extractfor reprogramming: One reprogramming reaction contains 250 ul extract(to 50-100K cells)

Prepare ATP generating system, keep on ice: mix ATP, GTP, creatinekinase, phosphocreatine in 1:1:1:1 ratio, keep on ice. Add 12.5 μl ATPgenerating system per reaction to the extract. Add 250 μl salmon eggextract (with ATP-generating system). Make sure the extract covers thecells on coverslips. Mix by tilting the plate. Incubate in incubator 60min, tilt the plate every 10 min. Aspirate the extract (200 μl) and addCa-enriched medium to each well (ca 1500 μl). Incubate for 2 h. Check inmicroscope if cells have attached to coverslips. If so, removeCa-containing medium and add complete medium (ca 500 μl). Incubate 37°C., 5% CO₂. Assess cells within 24 hours culture. Phase contrastmicroscopy. Split cells before confluence is reached. Coverslips weremoved to new wells one day after reprogramming, and some coverslips weretrypsinated to be moved to small bottles. Since cells didn't loosen, theentire coverslip was moved to the bottle.

Results—hsF Reprogramming

Reprogramming experiments RPE (starvation media) and RPF (normal media).

Changes in Gene Expression:

TABLE 9 Fold up regulation of the developmentally regulated OCT4 andNANOG genes, relative to GAPDH, as assessed by qPCR. OCT4 NANOG RPE 1&2150.71 10.14

Morphological changes of cells. After reprogramming, cell cultures wereassessed by phase contrast microscopy and compared with normal cells.

One day after reprogramming, a population of the surviving cellsresembled normal hSF cells, while a subpopulation of cells displayedaltered morphology. These cells appeared longer/more stretched thannormal cells and some (especially those from starvation media) showedcircular vesicles/bodies in the cytoplasm. More cells survived from thestarvation media than from the normal media.

From day 12 to day 22 after reprogramming (experiment terminated on day22), cells that were still attached to coverslips showed an unusualmorphology, with a larger and more distinct nucleus having thin“offshoots”/“spurs” and cells having a different shape than normalcells. A subpopulation of cells (mainly starved cells) still hadcircular vesicles/bodies inside the cytoplasm. As complete reprogrammingof all cells in each experiment is not expected (Taranger et al., 2005),the subpopulation of cells showing altered morphology probably representreprogrammed cells which are responsible for the alterations of geneexpression detected by qPCR.

Immunofluorescence. Cells were fixed on coverslips on day 7 afterreprogramming. Immunofluorescence labeling was conducted basically aspreviously described for tissue sections (Boulland et al., 2004).Briefly, cells grown on coverslips were fixed in 4% PFA (30 min RT),washed in PBS, blocked with 1M ethanolamine, washed in 3×PBS,preincubated in block solution (1 hr RT), incubated with primaryantibodies against OCT 3/4 (Santa Cruz) (1:200) in incubation solution(3 hrs RT), rinsed in 3×PBS and incubated with fluorescence-coupledsecondary antibodies Alexa 488 (1:2000) (Molecular Probes)(1 hr RT) andfinally rinsed in 3×PBS. To stain nuclei, DAPI (1:1000) was added tosecond to last rinse. Coverslips were mounted with ProLong Gold Antifadereagent (Molecular Probes) and images were taken with a fluorescencemicroscope (Olympus) or confocal microscope (Zeiss).

OCT4 staining was seen in the cytoplasm of most cells, stronger labelingwas seen in the reprogrammed cells compared to the normal control, whichshowed very weak staining. Hoechst staining was observed in the nucleiof normal cells, additionally overlapping with OCT4 staining in thecytoplasm of the reprogrammed cells. The cells were assessed forinfection to ensure the cytoplasmic Hoechst stain was not caused bymycoplasma but rather a true expression of reprogramming.

Subculture of HEKa Cells (Human Epidermal Keratinocytes—Adult)

Keratinocyte Culture Systems from Cascade Biologics

Extended-Lifespan Systems

Basal Medium EpiLife® Medium

Growth Supplement HKGS (S-001-5)

Subculture Reagent Trypsin/EDTA (R-001-100)

Subculture Reagent Trypsin Neutralizer (R-002-100)

Antibiotics (after reprogramming) Gentamicin/Amphotericin B (R-015-10)

Expected lifespan from HEKa (C-005-5C) 35-45 population doublings

Complete Media for HEKa

500 ml EpiLife Medium

5 ml HKGS (Human Keratinocyte Growth Supplement)

After reprogramming: 1 ml Gentamicin/Amphotericin (GA)

Culture the cells in 75 cm2 culture flasks—ca 10 mill cells atconfluence.

Subculture HEKa Cells

1. Quickly rinse cells with 3 ml of Trypsin/EDTA.

2. Add 1 ml of fresh Trypsin/EDTA and incubate until the cells aredispersed (8-10 min).

3. Add 3 ml of Trypsin Neutralizer solution and transfer the cells to asterile 15 ml tube. Repeat with additional 3 ml additional TrypsinNeutralizer.

4. Centrifuge at 180×g for 7 minutes.

5. Resuspend the cell pellet and seed new culture vessels with 2.5×103cells/cm².

6. Change media on cells after 48 hours

7. Change the medium every other day until the culture is approximately50% confluent.

8. Change the medium every day until the culture is approximately 80%confluent.

Freeze Cell-pellet:

7. Make fresh freeze-media:

-   -   a. Normal media added 10% FCS and 10% DMSO

8. Follow the above protocol (subculture) to point 4.

9. Wash cells with PBS (180×g, 7 min)

10. Resuspend the cell pellet to 1 million per 1 ml freeze media and add1 ml to cryo tubes.

11. Freeze the cells in Mr. Frosty-box with isopropanol at −80° C. overnight (−1° C./min).

12. Transfer to nitrogen tank.

Make Pellets for RNA-Isolation:

Follow the subculture protocol to point 4; thereafter

8. Resuspend cells to 1 million per 1 ml PBS and add 1 ml to eppendorftube for RNA pellets.

9. Spin at 300×g 10′ at 4° C.

10. Aspirate PBS, keep pellet on ice and snap-freeze in liquid N2.

11. Transfer to −80° C.-freezer.

Reprogramming of HEKa Cells (without SLO)

Objective: To reprogram cells with a nuclear-free extract to alter geneexpression, morphology and elements of growth and to study changes instate of differentiation.

HEKa cells used for the experiment were grown in their normal media(EpiLife with HGKS and alternatively 1% GA [after reprogramming]).“Mock” reprogramming was conducted as control (cells undergoreprogramming procedure in normal media without extract added) andnormal HEKa cells were cultured in parallel as negative control.

293T were reprogrammed in the same experiment, also including one mockreprogramming and 1 flask of normal 293T as controls. 293T are grown intheir normal media (RPMI with 1% PS).

Materials: 1 flask HEK cells; 1 flask 293T; Extract (salmon eggextract); Incubation in media for control; RPMI medium (293T); EpiLifeMedium (HEK); 1×PBS; Ca2+ free Hanks Balanced Salt Solution (HBSS) at 4°C.; TE to loosen HEK cells from flask; TN (Trypsin Neutralizing)solution; ATP (200 mM stock in water); GTP (10 mM stock in water);Phosphocreatine (2M stock in water); Creatine kinase (5 mg/ml stock inwater); NTP (25 mM stock); Autoclaved mq water; 75 cm2 flasks; 15 ml,1.5 ml tubes Centrifuge cooled to 4° C.; Swing out bucket rotor for 1.5ml tubes, and for 15 ml tubes; Waterbath at 37° C.

Procedure:

1. Harvest HEK cells—wash with 1 ml TE, aspirate and incubate with 3 mlTE for 5-10 min Transfer cells into 15 ml tubes and spin at 200×g, 10min 4 C. (Harvest the 293T cells—wash with PBS, add 10 ml RPMI media,loosen cells and transfer to 50 ml tube).

-   -   a. Wash once in 30 ml ice cold PBS and one in 10 ml ice cold        HBSS    -   b. Resuspend cells to 500.000 per 1 ml HBSS

2. Add 500 000 cells into each reprogramming tube

-   -   a. Spin 1200 rpm, 5 min 4° C. in SW rotor    -   b. Remove HBSS

3. Prepare extract for reprogramming

-   -   a. Prepare ATP generating system, keep on ice: mix ATP, GTP,        creatine kinase, phosphocreatine in 1:1:1:1 ratio, keep on        ice.+0.5 mM NTP per reaction    -   b. Add 30 μl ATP generating system per reaction

4. Add extract (with ATP-generating system), 500 ul to 500.000 cells pertube.

-   -   a. Cover tubes with parafilm and incubate for 60 min in        waterbath at 37° C. Flick cells twice during incubation.

5. Add one reprogramming tube per flask with medium.

6. Incubate 37° C., 5% CO2

Assess cells within 24 hours culture. Phase contrast microscopy. Splitcells before confluence is reached.

Results: HEKa Reprogramming

Reprogramming experiment RPH.

Changes in Gene Expression:

TABLE 10 Fold upregulation of the developmentally regulated OCT4 andNANOG genes, relative to GAPDH, as assessed by qPCR. OCT4 NANOG RPH mockd13 1.29 1.26 RPH3 d13 4.13 13.45Morphological Changes of Cells:

After reprogramming, cell cultures were assessed by phase contrastmicroscopy and compared with normal cells.

Fewer cells incubated with extract survived the reprogramming procedurethan control mock cells. Some reprogrammed cells showed circularvesicles/bodies inside the cytoplasm and possibly larger and lessdefined nuclei. Some of the cells showed an atypical morphology withsmall “spikes” resembling podocytes protruding from the plasma membraneand having a different overall shape than normal cells.

Immunofluorescence

Cells were fixed on coverslips on day 9 after reprogramming.Immunofluorescence labeling was conducted as described for hSF cells.

HEK cells were immunolabeled for OCT4 (same procedure and antibody asfor hSF cells) and nuclei identified by Hoechst stain in a fluorescentmicroscope. Normal HEK cells showed very weak OCT4 staining in thenuclei, while nuclei of a subpopulation of HEK cells reprogrammed withsalmon egg extract were clearly more strongly stained with OCT4, inagreement with qPCR results showing an upregulation of OCT4 in cellsfrom the same reprogramming. Hoechst staining was overlapping with OCT4staining in the nuclei of most cells, and OCT4 staining was especiallystrong in the nucleoli of the reprogrammed cells. Negative controls(primary antibody omitted) showed no OCT4 staining while nuclei werenormally stained by Hoechst. Identical settings for microscope anddigital image capture were used when observing cells.

Example 13 Preparation of Salmon or Trout Egg Extract

This example describes the preparation of LEX extracts from fresh salmonor trout roe/eggs sent on ice overnight from hatchery. Eggs that havebeen in transit on ice >48 hours are discarded. If the eggs can't beprepared on the day of arrival, the eggs may be stored at −20° C. for upto 12 months. The extracts are prepared using an Avanti J-26 XPultracentrifuge with a JLA 8.1000 rotor and 6×1000 ml tubes(polypropylene, #363678 with liner).

The day before preparation of extracts, the glass and stainless steelequipment is autoclaved. Ten liters PBS/0.9% NaCl are prepared andplaced in a cold room. Five liters of Buffodine (50 ml in 5l 0.9% NaCl)is prepared.

On the day of extraction, the rotor is pre-cooled by placing the rotorin a centrifuge with all 6 cannisters and programming the centrifuge(speed 2000 g, time 30 min, temp 4° C. and “start”). The followingequipment is placed in the cold room: metal potato masher, metal sieves,funnels, tweezer(s), 3 L, 1 L, and 500 ml glass-beakers, (tubes: sterile(autoclaved) eppendorf tubes, 50 ml tubes/canisters, 200 ul tube strip,Buffodine and ice-cold NaCl for washing, sea salt for washing equipment,ice boxes for preparation, sterile needles (14 G) & syringes (50+ ml)for removing extract from centrifuge tubes. Liners are also placed inrotor buckets in rack.

The materials are handled in a cold room and eggs, homogenate andextract on are kept on ice at all times. The eggs are washed inbuffodine for 10 minutes (1:100 Buffodine in 0.9% NaCl) and drained. Theeggs are rinsed 4× in NaCl. The sieve with eggs drained well betweenwashes. Eggs are homogenized by crushing in metal potato masher directlyinto glass beaker through sieve placed on funnel (work quickly and incold room. Egg shells and debris are discarded from sieve and masher(all extract trash in biowaste). The homogenate is transferred tocentrifuge tubes by pouring through funnel into liners in tubes. Allbucket are weighed to assure that the maximum weight difference is <30g. Buckets and then closed and placed in canisters in the rotor. Thehomogenates are centrifuged 1 hour in JLA 8.1000 rotor at 7.000 rpm(12200 RCF (g)) at 4° C. The tubes are removed following centrifugationand the middle (cytoplasmic) fraction is collected by inserting a needleabout 0.5 cm over bottom of the tube. Care is taken not to pollutefraction with top lipids (the top fraction) or bottom debris. Anymaterial containing lipids or debris is discarded. The middle fractionis transferred directly to freeze-resistant containers (microfuge tubes(1 ml aliquots), 8-strip PCR tubes (200 μl aliquots) and 50 ml tubes orother canisters. About 1 ml is retained on ice for testing. Extractsaliquots are frozen immediately at −80° C. and can be stored for up toone year. The extracts have a pH of from 6.5-7.0; have a bacterial loadof less <100 colonies per ml (e.g., <10 colonies per plate) as tested onantibiotic-free agar plates; an osmolarity of from 300-500 mOsm; and aprotein content of from 100-300 mg/ml.

Example 14 Egg Extracts Increase Collagen Production

Egg extracts prepared as described in Example 13 were applied tofibroblasts in vitro and collagen production was assayed. Briefly, onday −1, fibroblasts were seeded in cell culture flasks. On day zero, theculture media was replaced with fresh culture media supplemented with0.5% LEX. Control cells were cultivated with cell culture medium withoutLEX. The cultures were continued for seven days, with the mediasupplemented with LEX changed every 24 hours. On day 8, the cells werewashed 3× with PBS and low serum cell culture medium was added. On day9, the culture media was harvested and collagen content was assessedusing a collagen kit (Bicolor). Cells cultured in the presence of mediumsupplemented with LEX (four different preparations) demonstrated astatistically significant increase in collagen production. The data issummarized in the following table.

TABLE 11 Collagen content increase in media (%) using different batchesof LEX LEX batch % increase over control P value LEX6 1800%  LEX18 532%0.01 LEX19 617% 0.001 LEX20 547% 0.01

Example 15 Stimulating Cells with Extracts for 8 Days, then for aFurther 7 Days without Extract (Total 15 Days) Show that the Effects ofthe Extract on Collagen Secretion are Reversible

This experiment shows that the effect of treating cells with the extract(LEX) for 8 days (giving a 367% increase in collagen production at day8) was significantly reduced when treating the cells for another 7 dayswith cell culture medium without extract (50% increase remained). Thisindicates that the effect of the extract on the fibroblasts isreversible.

Example 16 Egg Extracts Increase Fibroblast Proliferation

Egg extracts prepared as described in Example 13 were applied tofibroblasts in vitro and collagen production and proliferation wereassayed. Briefly, on day −1 fibroblasts were seeded in cell cultureflasks. On day zero, the culture media was replaced with fresh culturemedia supplemented with 0.5% LEX. Control cells were cultivated withcell culture medium without LEX. The cultures were continued for fivedays, with the media supplemented with LEX changed every 24 hours. Cellnumber was determined in the flasks. Cells cultured in the presence ofmedium supplemented with LEX (two different preparations) demonstrated astatistically significant increase cell number. The data is summarizedin FIG. 5.

Example 17 Comparison of Trout Roe, Fertilized Salmon Roe, andUnfertilized Salmon Roe

Fertilized salmon egg extracts, unfertilized salmon egg extracts, andtrout roe extracts prepared as described in Example 13 were applied tofibroblasts in vitro and collagen production and proliferation wereassayed. Briefly, on day −1 fibroblasts were seeded in cell cultureflasks. On day zero, the culture media was replaced with fresh culturemedia supplemented with 0.5% LEX. Control cells were cultivated withcell culture medium without LEX. The data is summarized in FIG. 6.

Example 18 Egg Extract Enhances Absorption into Skin

Salmon egg extracts prepared as described in Example 13 wereincorporated at a final concentration of 4% in a salve (Trial 1) orcream (Trial 2). The test treatments were applied to one hand while acontrol treatment lacking the egg extract was applied to the oppositehand. Five test persons participated in each trial for one week. Thetest persons filled out a survey following the treatment. The resultsare presented in Table 12.

TABLE 12 % preferring extract TRIAL 1 TRIAL 2 Questions answered:(right) (right) 1 Noted a difference left/right? 100 100 2 What type ofdifference noted (for cream with LEX) 1. better absorption 100 100 2.softer skin 100 100 3. effect on small wounds 25 25 4. effect onwrinkles 25 25 3 Visible improvement in skin treated w/LEX 100 50 after7 days 4 Preferred cream (% preferring extract of 100 100 total withpreference) 5 Noted difference in smell or texture of 100 50 creamsTRIAL 1 Vitapan salve w/4% manuka honey and 4% LEX2 (strong effect oncells). All test persons noted a clear difference. TRIAL 2 Vitapan creamw/4% LEX13 (good effect on cells). Some test persons noted a cleardifference, others a moderate difference.

Example 19 Physical Properties of Extracts

RNA, DNA and protein content of LEX were measured using the Qube-iTfluorimeter from InVitrogen. All extracts measured have yieldedcomparable effects on collagen secretion from human fibroblasts in vitroat 0.5% stimulation for 8 days. Extracts were diluted in PBS and Qube-iTassay buffer prior to measurements.

RNA content of salmon and trout homogenates and extracts average 2-5mg/ml. Homogenates of salmon eggs (non-centrifugated) contain 3-4 mg/mlRNA After centrifugation to 9-15,000 g, RNA content was reduced to 2-3mg/ml. This is probably due to RNA being centrifugated down or degraded.Interestingly, trout egg homogenates (non-centrifugated) contain 2-3mg/ml RNA, but after centrifugation to 9-15,000 g, the concentration ofRNA is increased to 3-5 mg/ml. Extracts made from trout eggs are lessviscous than extracts made from salmon eggs, and may keep RNA better inwater phase suspension during centrifugation.

DNA content of salmon and trout homogenates and extracts between 40-500ng/ml. Homogenates of salmon eggs (non-centrifugated) contain 60-200μg/ml DNA After centrifugation to 9-15,000 g, DNA content was reduced to40-51 μg/ml. This is probably due to DNA being centrifugated down.Interestingly, homogenates of trout eggs (non-centrifugated) containmore DNA than salmon egg extracts: 130-530 μg/ml DNA. Aftercentrifugation to 9-15,000 g, DNA content is reduced to 70-125 μg/ml,but is still higher than comparable salmon egg extracts. Extracts madefrom trout eggs are less viscous than extracts made from salmon eggs,and may keep DNA in better water phase suspension during centrifugation.

The DNA content varies widely between test-homogenates prepared here,and may be caused by differential lysing of nuclei containing gDNA priorto centrifugation. Better lysing of nuclei by variations on thehomogenization process during production may yield extracts with higherDNA content. These differential extracts may yield separate effectsuseful for different applications, such as effects on gene expression inskin cells.

Protein content of salmon and trout homogenates and extracts average180-300 mg/ml. Homogenates of salmon eggs (non-centrifugated) contain180-260 mg/ml protein. After centrifugation to 9-15,000 g, proteincontent was unchanged or increased slightly to 200-260 mg/ml.Homogenates of trout eggs (non-centrifugated) contain 250-300 mg/mlprotein, and after centrifugation to 9-15,000 g, protein content isroughly the same (250-270 mg/ml). The protein fraction of the eggcytosol is not expected to be spun down at the g-forces applied, and maybe expected to be similar to the raw protein content of the egg cytosol.

Previous measurements of protein contents in extracts using a Nano-dropspectrophometer showed a range of 150-250 mg/ml. This may be due to anupper detection limit around 250 mg/ml in the Nano-drop. It is probablethat the slightly higher fluorometer measurements presented here aremore accurate.

TABLE 13 Summary of measurements RNA, DNA and protein content inextracts Source Centrifugation LEX/corresp mg/ml μg/ml mg/ml of eggsspeed to LEX RNA DNA protein Salmon Homogenate, no LEX20 3.51 66.8 256centrifugation Salmon 15000 xg LEX20 2.34 44 252 Salmon Homogenate, noLEX24 3.42 192.4 180 centrifugation Salmon 12000 xg LEX24 2.93 50.8 208Trout Homogenate, no LEX28 2.67 131.6 249 centrifugation Trout 15000 xgLEX28 3.51 73.2 249 Trout Homogenate, no LEX25 2.53 528 296centrifugation Trout 15000 xg LEX25 3.70 72.8 262 Trout 15000 xg LEX253.63 99.2 210 Trout 12000 xg LEX31 4.59 87.2 270 Trout 12000 xg LEX324.68 124.8 — Trout 12000 xg LEX33 4.67 94.4 252

Lipid Content of extracts is 3.7-4.5 g/100 g extract (3.7-4.5%). Thelipid content of extracts were measured by ALS (Germany), and was foundto be in the narrow range of 3.7-4.5 g/100 g in all extracts from salmonor trout roe prepared at centrifugations spanning from 1,700 g to 15,000g. The lower g-force centrifugations appear to require spinning at roomtemperature to give equal lipid fractionation to higher g-forces at 4degrees centigrade.

Summary of physical properties. Preparation of extract from homogenatesof salmon and trout eggs give differential separation of RNA, DNA andprotein, but equal separation of lipids. Fertilized and unfertilizedsalmon egg extracts display the same profiles of protein, RNA and DNA.

1) The protein concentration (180-300 mg/ml) of the extract is roughlycomparable to that of the homogenate (no or little protein removed byproduction method regardless of g-force).

2) The RNA content (2-5 mg/ml) seems roughly equal for salmon and trouthomogenates, slightly lower in trout. RNA seems to be increased in theextracts made from trout egg homogenates, which may be due to the lesserviscosity and better solubility of RNA in the extract fraction fromthese eggs. RNA content in final salmon egg extracts is slightly lowerthan that of trout egg extracts.

3) The DNA content (40-500 ug/ml) of the extracts is highly variable,which is probably caused by differential lysing of nuclei in theegg-crushing homogenization process. Salmon egg extracts appear to havelower DNA content than trout egg extracts. In both extracts, and DNAcontent is lower in extracts than in homogenates, indicating that someDNA is spun down at g-forces over 9.379 g.

4) Total lipid content (3.7-4.5%) is roughly equal for salmon and troutextracts. It seems equal amounts of the lipids are separated from theextract fraction at most g-forces over 1700 g.

Example 20 Production of Extracts

It has been documented that lipid content of the extract surprisingly isunchanged at centrifugation speeds varying from 1,700 g to 15,000 g (seeabove), while other parameters such as RNA, DNA and protein content isaltered with the increase of g-force during centrifugation.

An extra step of washing the eggs for 10 minutes with buffodine (1:100in 0.9% NaCl) before preparation of homogenate is beneficial. Thiswashing step appears to reduce the bacterial content significantly. Forsafety reasons, all LEX batches packaged in final containers are mildlypasteurized (incubated) by heating to 56° C. for 20 minutes. Thispasteurization sterilizes the extract completely, with 0 bacteria foundin extracts plated on bacteria dishes incubated for 3 days at roomtemperature, 4 degrees centigrade or 30 degrees centigrade. 1 colony/100μl LEX plated on agar dish incubated at room temperature is the maximumobserved. This is 100× below safety limits for drinking water (100bacteria/ml). A single colony seldom observed probably comes from theair during the plating of LEX, and is comparable to bacterial growth ofnegative control (plate only).

The stability of LEX and collagen secretion effect is retained after LEXis heated to 56 C for 20 minutes. When applied to human fibroblasts invitro at 0.5% concentration in cell media for 8 days (media changeddaily), the effect on collagen secretion (as measured as efflux ofcollagen from cells to cell medium and compared to untreated controlcells), was comparable to cells treated with unheated extract which hadbeen kept at −80 C after preparation. A 200-400% increase compared tocontrols was observed for both heated and unheated LEX. Previously wehave seen a decreased effect on collagen secretion with extractsincubated at 72 C, indicating that active substances in the extractswhich may be denatured between 56 and 72 C are responsible for parts ofthe secretion effects. In this temperature range, proteins are known todenature. It may be deduced that a structured protein is one of theactive substances.

Example 21 Extracts Increase Collagen Production in Humans In Vivo

This Example describes the effects on collagen gene expression andprotein content on intact skin after 14 day application of 2% LEX incosmetic cream.

Experimental design: Two healthy volunteers (2 male, here labeledIndividual 1 and 2, respectively) applied a skin cream to left upperarm, and the same cream with 2% LEX to right upper arm 2 times daily for14 successive days. No other cream products were used on skin under theduration of the study. Each individual is thus their own negativecontrol. This as collagen amount in skin is deemed to be different inthe individuals.

Upon completion of the 14 day treatment, 3 mm punch biopsies of fullskin thickness were taken from the treated areas, left and right upperarm respectively, as well as a equal biopsy from the lower arm asuntreated control. A total of 3 biopsies were taken from each of the 2persons, and labeled by random number by a dermatologist. The studiedwas blinded and the scientists conducting the evaluation were only givenaccess to the numbers coding for the different biopsies after completionof experiments and collection of raw data.

The three biopsies from each individual are labeled: LEX (cream with 2%LEX), cream (cream alone), control (untreated skin). The biopsies werefrozen in liquid Nitrogen after excision and stored at −80 C before RNAand protein isolation. Protein and RNA were isolated simultaneously frombiopsies by using a modified method of the RNeasy micro kit from Qiagen.RNA was used for cDNA synthesis, and qPCR was performed with primers forGAPDH (reference gene) and Collagen I, using SybrGreen reagents andBioRad RealTimePCR machines.

Protein pellets isolated from the biopsies were dissolved in PBS withand without 5% SDS and total protein concentration of each sample wasmeasured using the Quant-iT Protein Assay. The amount of collagen in thesame protein samples was measured by a collagen assay and measured in aspectrophotometer (Nano-drop). The relative amount of collagen comparedto total protein in each sample was calculated from these measurements.(Note that protein pellets were difficult to dissolve in assay buffers,which may have influenced the final results. As the final result isgiven as a ratio (relative number) of the 2 measurements on individualsamples, this may resolve the issue as it may be assumed that eachsample is equally dissolved. Addition of SDS improved solution of theprotein.)

Results of Gene Expression as Measured by qPCR:

TABLE 14 Fold up regulation of collagen I gene in skin biopsies LEXtreated Cream treated Untreated Individual biopsy biopsy control 1 1.7x0.6x 1 2   6x 2.5x 1

Individual 1:

1.7× increase in collagen RNA in LEX treated skin.

0.6× decrease in collagen RNA in cream treated skin.

Individual 2:

6× increase in collagen RNA in LEX treated skin.

2.5× increase in collagen RNA in cream treated skin.

Discussion of gene expression: The results showed that 2 of 2individuals had 2.5-6 fold up-regulation of the collagen I gene in skintreated with cream with 2% LEX compared to untreated control skin. Thecollagen gene expression in skin treated with cream with 2% LEX was forboth individuals much higher than in skin treated with cream alone. Thecream alone yielded a low but insignificant decreased collagen geneexpression compared to untreated control skin in one individual, while aslight increase in the other two.

Conclusion: Using cream alone has little or no effect on collagen geneexpression, while the same cream with 2% LEX increases gene expression1.7-6 fold. The final conclusion is proof of concept that 2% LEXincreases collagen I gene expression in human skin in vivo after 14 daysof treatment. This is in agreement with data from our studies on humanskin fibroblasts in vitro, where 0.5% LEX-treatment for 8 days gave upto 6-fold increase of Collagen I gene expression.

Results—collagen protein content: Protein concentration measurementsvaried between samples dissolved with/without SDS. As it is impossibleto say which measurement is superior, an average of both measurements oftotal protein concentration and collagen I concentration was used forcalculating ratios. Measurements showed that the individual biopsies hadyielded different amounts of protein, thus a relative value of collagencontent/total protein for each sample was calculated to correct fordifferent protein isolation efficacy. The ratio found for the untreatedcontrol biopsy was then set as 1 for each individual, to compare ratioof collagen/total protein:

TABLE 15 Relative amount of collagen compared to total protein contentextracted from each biopsy. Ratios collagen/total protein for eachbiopsy. Control biopsy is set as 1 for each individual. LEX treatedCream treated Untreated Individual biopsy biopsy control 1 1.3 0.4 1 21.3 0.9 1

Discussion of collagen protein content: The results show that bothindividuals with a full set of skin biopsies had the highest relativeamount of collagen/total protein in the biopsy treated with 2% LEX incream. The amount of collagen being increased by 1.3× compared tountreated control skin. Interestingly, for all 3 individualscollagen/total protein ratio decreased in skin treated with cream alonecompared to untreated control skin. The cream may contain agents thatincrease other substances in the skin, drowning the amounts of collagensecreted. The indication that LEX added to the cream may increasecollagen gene expression, may imply that the cream itself may reducecollagen secretion, making it possible that another cream should be usedfor addition of LEX.

When comparing results from qPCR and protein measurements, thealterations in gene expression and relative protein concentration foreach biopsy sample was remarkably similar. This strengthens the resultsfurther, showing the alterations in collagen in LEX treated skin on bothgenetic and protein expression.

TABLE 16 Fold regulation of collagen gene expression (gene) andcollagen/total protein ratio (protein) in human skin biopsies in vivoafter 14 day treatment of cream w/wo LEX LEX Cream Untreated treatedbiopsy treated biopsy control Individual protein gene protein geneprotein gene 1 1.3 1.7 0.4 0.6 1 1 2 1.3 6 0.9 2.5 1 1When taken together, the study shows that addition of 2% LEX to acosmetic cream increases expression of the collagen I gene and therelative amount of collagen to total protein. The results also show thatusing the cream alone has no, or even negative, effects on collagencontent in human skin in vivo. In conclusion, it is the addition of 2%LEX which gives the effect on collagen gene and protein expression.

Example 22 Results from Human In Vivo Wound Healing Study

This Example describes the effects on wound healing after a 14 dayapplication of pure, concentrated LEX to 3 mm diameter punch biopsywound.

Improved wound healing by treatment of pure LEX: 3 mm diameter punchbiopsies of full skin thickness were taken on a human subject, 1 on eachupper arm and 1 on each lower arm, 4 biopsies total. The wounds were ofequal size and depth.

Biopsies on right arm (upper and lower) were used as controls(untreated), while biopsies on left arm were treated 1× daily witheither LEX32 (wound on upper arm) or Elisabeth Arden 8-hour cream (woundon lower arm). The biopsy wounds were covered with band-aids the first 8days until scab formation. Band-aids were changed 1× daily, at time ofapplication of LEX/8-hour cream. After scab formation, the wound waskept uncovered, and LEX was applied to the wound daily until day 28.Pictures of each wound and scar were taken daily with a digital cameraand a millimeter ruler held next to wound to compare wound sizes duringthe healing process.

LEX of batch 32 was used for this study. The batch was made from trouteggs and bacteria free. QC was within normal range of typical LEXproduct. The extract was stored in −20 C and 1.5 ml tubes thawed on day1, day 5, day 10 and day 20. A small amount of LEX was applied withq-tip directly to wound, and the thawed tube stored at 4 C for up to 10days. No smell developed, and there were no sign of infection orbacterial growth in LEX nor wound.

Summary of Results:

TABLE 16 8-hour cream treated Untreated Day LEX-treated wound positivecontrol wound control 0 3 mm biopsy taken 3 mm biopsy taken 3 mm biopsytaken 3 Wound dry Open wound wet Open wound wet 5 Wound dry, thin Wounddry w/scab. Wound dry with scab, visibly better Same size as scab. Samesize as contracted/smaller untreated control. 8-hr cream. than controlsRedder than control. 13 Wound ⅔ size of Red scarring around Red scarringaround control wound, less edges, large scab edges, same size as red andwith 8-hour cream, smallest scab smaller scab 18 ReepithelializationScab, red swollen Scab, red edges Scab off, scar light edges pink witheven parameter 21 Scar flattened and ReepithelializationReepithelialization light pink Scab off, scar redder, Scab off, drywhite more uneven and flakes under scar, swollen than LEX and red edgesuntreated control 28 Scar remodeling Scar remodeling Scar remodelingScar visibly smaller Scar red and Scar red, uneven with less thickeningthickening in the edges, thickening in in the middle middle. Tangiblethe middle. lump.

Discussion: The results presented in table format above indicate thatdaily application of pure 100% LEX speeds and improves wound healing onmany parameters. 8-hour cream (positive control) was not better, orslightly worse, than untreated on most parameters:

Faster drying of wound. The wound treated with LEX was dry (noextracellular fluid oozing from wound) on day 4, a full day before theother 2 wounds. This may be partly because the LEX itself dries andforms a dry membrane, protecting the wound. A complete dry scab alsoformed first among the 3 wounds.

Faster reepithelialization. Wound treated with LEX lost scab (defined asreepithelialization) 3 days before untreated wound or wound treated with8-hour cream (day 18 compared to day 21).

Reduced inflammation. Throughout the healing process, the wound treatedwith LEX was less red, less puffy and looked less inflamed than theuntreated wound, and particularly better on these aspects than the woundtreated with 8-hour cream, which was puffy, red and itchy beforecomplete reepithelialization. The lesser redness of the LEX treatedwound was particularly visible from day 5-23. The fluid LEX applied tothe wound contains a high concentration of proteins and also marinesalts. The LEX dries to a film on the wound, and may protect it fromirritants and air borne pathogens. The 8-hour cream is very sticky andthick, making pathogens and clothing fibers etc. stick, irritating thewound, and may thus increase risk of inflammation in wound. The woundtreated with 8-hour cream appeared worse than the untreated wound on allparameters from the day the band-aid was removed (day 8) until the daythe scab fell off (day 21).

Faster wound contraction. The LEX treated wound started to retractaround the edges on day 3, compared to day 6 in the other wounds.Particularly striking, was how the wound edge appeared different in thetreated wound: The edge retracted toward the bottom of the wound,forming a menisca along the bottom edge, as if the wound was closingfrom below. The other wounds appeared to close by lateral contraction,where the depth incision of the wound remained antiparallel to thebottom of the wound for the first 10 days. The better contractionnoticed in the LEX treated wound may be due to faster differentiation offibroblasts to myofibroblasts and faster movement of these into thewound from the wound edges.

Earlier and better remodeling. After a wound is scab free and the scarvisible, remodeling of the scar starts by reorganization of collagenfibers within the scar and activation of differentiation processesbetween fibroblasts and myofibroblasts. Myofibroblasts responsible fordepositing collagen during the wound closure process leave the scar, andnormal fibroblasts deposit type I collagen while a machinery of enzymesbreak down and build up collagen fibers in different directions in thewound. Blood vessels also rearrange, and inflammatory processes recede.As the inflammatory process decreases and collagen fibers arereorganized in plane with the skin, the scar flattens. The scar from theLEX treated wound was visibly flatter from the day the scab fell off(day 21), and proceeded to look better than the other 2 scars throughoutthe remodeling process.

Smaller and less red scar with better visual appearance. From the daythe scab fell off (day 21) the LEX treated scar appeared less red,smaller and flatter than the other scars. In combination, each of theseparameters makes the scar less visible and more healthy looking.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention that are obvious to those skilled in cellbiology, or molecular biology or related fields are intended to bewithin the scope of the following claims.

What is claimed is:
 1. A method of improving wound healing in a humansubject, comprising: applying a composition comprising a trout eggcellular extract having a protein content of from about 210-270 mg/mland an RNA content from about 3.51 to 4.68 mg/ml to a wound of saidsubject under conditions such wound healing is improved, wherein saidimprovement is selected from the group consisting of faster drying,faster reepithelialization, reduced inflammation, faster contraction,earlier remodeling, reduction in scar tissue and improved visualappearance of the wound and combinations thereof.
 2. The method of claim1, wherein said composition is applied to a wound in the skin of saidsubject.
 3. The method of claim 1, wherein said composition is providedin a cream, gel, emulsion, ointment, spray, powder or lotion.
 4. Themethod of claim 1, wherein said trout egg extract is a cytoplasmicextract.
 5. A method of improving wound healing in a human subject,comprising: applying a composition comprising a salmon egg cellularextract having a protein content of from about 180-260 mg/ml to a woundof said subject under conditions such wound healing is improved, whereinsaid improvement is selected from the group consisting of fasterhealing, increased scar tissue reorganization, less visible scar tissuethickness, lower epidermal thickness, lower dermal thickness andcombinations thereof.
 6. The method of claim 5, wherein said compositionis applied to a wound in the skin of said subject.
 7. The method ofclaim 5, wherein said composition is provided in a cream, gel, emulsion,ointment, spray, powder or lotion.
 8. The method of claim 5, whereinsaid salmon egg extract is a cytoplasmic extract.